j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Prognostic value of biomarkers in metastatic colorectal cancer patients Kozo Kataoka, MD,a,* Akiyoshi Kanazawa, MD, PhD, FACS,b Akio Nakajima, MD, PhD,a Ayane Yamaguchi, MD,a and Akira Arimoto, MD, PhDa a
Department of Surgery, Osaka Red-Cross Hospital, Osaka, Japan Department of Gastroenterological Surgery and Oncology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan
b
article info
abstract
Article history:
Backgrounds: The prognostic value of biomarkers in metastatic colorectal cancer (mCRC)
Received 29 July 2014
patients with liver metastases remains unclear. We assessed the difference of expression
Received in revised form
of biomarkers between primary tumors and liver metastases treated with chemotherapy in
9 September 2014
mCRC patients, as well as the prognostic value of these markers.
Accepted 3 October 2014
Methods: Forty-three mCRC patients with liver-limited disease from January 2007
Available online xxx
eNovember 2011 were analyzed. They all received resection of primary tumors followed by oxaliplatin-based chemotherapy. After chemotherapy, they all received hepatic resection.
Keywords:
Forty-three paired primary and metastatic tumor specimens were collected to measure the
Colorectal cancer
messenger RNA expression of six biomarkers by the Danenberg tumor profile method
Liver metastases
(thymidylate synthase, dihydropyrimidine dehydrogenase [DPD], excision repair cross-
Prognostic marker
complementing gene1, thymidine phosphorylase [TP], folylpolyglutamate synthase, and
TP
regenerating islet-derived family, member 4).
DPD
Results: Thirty-six patients’ messenger RNA was used for analysis. All markers showed similar expression between primary and metastatic sites. The low-expression group of Danenberg tumor profile and TP in the primary tumor showed significantly higher overall survival than the high-expression group (P < 0.001 and P ¼ 0.033), but for DPD and TP in liver metastases, there were no significant differences of overall survival between the two groups. The ratios of marker expression in liver metastatic site to that in primary site of DPD and TP were significantly higher in chemo-responders than in non-chemo-responders (P ¼ 0.034 and P ¼ 0.022). Conclusions: Biomarkers’ expressions in liver metastases were similar to those in the primary tumor. DPD and TP in the primary lesion may be a prognostic factor in chemotherapy-naı¨ve mCRC patients with liver-limited disease, but those in liver tumor were not. Further validated analysis to our results would be warranted. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Osaka Red-Cross Hospital, 5-30, Fudegasaki, Tennoji, Osaka 543 8555, Japan. Tel.: þ81 6 6774 5111; fax: þ81 6 6774 5131. E-mail address: [email protected] (K. Kataoka). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.006
2
1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Introduction
Colorectal cancer is one of the most common causes of cancer-related mortality worldwide, and the liver is the most common, and often the only, metastatic site [1,2]. Surgical resection of colorectal liver metastases (CLM) is considered the only curative therapy, but most metastatic colorectal cancer (mCRC) patients with CLM have unresectable disease [3,4]. It is important to develop the strategy of chemotherapy based on the expression of biomarkers to improve the survival of CLM patients. One of the concerns about biomarkers for CLM patients is to reveal the differences of marker expression between primary lesion and liver metastatic lesion. Some reports have described that the messenger RNA (mRNA) expression levels of several biomarkers (thymidylate synthase [TS], dihydropyrimidine dehydrogenase [DPD], thymidine phosphorylase [TP], and others) in the primary site and the liver metastatic site were similar or showed a positive correlation if laser-captured microdissection was used [5,6]. However, the association of expression of these markers between primary tumor site and metastatic site is not fully understood. In this study, we selected six specific genes as follows: TS, DPD, TP, excision repair cross-complementing gene 1 (ERCC1), folylpolyglutamate synthase (FPGS), and regenerating isletderived family, member 4 (REG4). TS, DPD, and TP are involved in the metabolism of fluoropyrimidines, and there are many reports about these markers in colorectal cancer [7e17]. ERCC1 is an excision nuclease within the nucleotide excision repair pathway that plays a major role in repairing platinum-induced DNA adducts [18]. There are some reports that ERCC1 might
predict the efficacy of oxaliplatin-combined chemotherapy and that expression of ERCC1 was associated with prognosis of colorectal cancer, but their predictive and prognostic value is still unclear [11,16,19]. Folate-metabolizing enzyme FPGS is involved in the metabolism of folic acid [14] and REG4, which is a member of the REG family that acts as an antiapoptotic factor through the AKT signaling pathway [20,21]. Using these biomarkers collected from the same patients’ primary tumor tissues and liver metastatic tissues, we assessed the difference of their expression between primary site and liver metastatic site treated with chemotherapy in CLM patients retrospectively. We also assessed the prognostic value of markers collected from two different sites of the same patient.
2.
Methods
2.1.
Patients
Forty-three mCRC patients with resectable liver-limited disease from January 2007eNovember 2011 were assessed. mCRC patients who had extrahepatic disease and who had unresectable liver metastases were excluded from the analysis. Resectability was decided based on the size of the remnant liver volume (>30%) and expected function after the removal of all metastases, regardless of the number and size of the liver metastases. If metastases infiltrated (1) all hepatic veins, (2) both hepatic arteries and 3 both portal vein branches, these patients were defined as unresectable. Patients who received chemotherapy within 12 mo of diagnosis of mCRC with liver-limited disease were also excluded from the analysis.
Fig. 1 e Patients’ flow. LLD, liver-limited disease. (Color version of the figure is available online.)
3
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 1 e Patients’ characteristics. 2007.1e2011.11
n ¼ 36
Median (range) Men Women T1, 2 T3, 4 N0 N (þ) Rectum Colon Synchronous Metachronous Median (range) Median (range) mFOLFOX6/Xelox mFOLFOX6/Xelox þ bevacizumab mFOLFOX6/Xelox þ cetuximab Median (range) Yes No CR, PR SD PD NE Median (range) 0e1 2
66 (27e81) 23 13 3 33 11 25 11 25 15 21 2 (1e11) 2.6 (0.9e12) 27 6 3 6 (3e22) 28 8 13 14 7 2 9.8 (1.5e1215) 36 0
Variables Age (y) Sex T category of the primary cancer Lymphatic spread of the primary cancer Location of primary tumor Interval to metastasis Number of liver metastases Size of largest liver metastasis (cm) Preoperative chemotherapy
Cycles of chemotherapy Adjuvant chemotherapy Clinical response to chemotherapy
Serum CEA level (ng/mL) ECOG performance status
CR ¼ Complete Response; PR ¼ Partial Response; SD ¼ Stable Disease; PD ¼ Progressive Disease; NE ¼ Not all Evaluated; CEA ¼ Carcinoembryonic antigen; ECOG ¼ Eastern Cooperative Oncology Group.
First, they underwent colorectomy if metastasis was synchronous. After that, they received oxaliplatin-based chemotherapy. The response to chemotherapy was evaluated using Response Evaluation Criteria in Solid Tumors criteria (version 1.1) [22]. After chemotherapy, they received hepatic resection. The interval between the last administration of chemotherapy and resection was generally about 28 d, and 42 d especially when bevacizumab was used. Tumor tissues were collected from both primary tumors and liver metastatic tumors. Paired mRNA expression levels of TS, DPD, TP, ERCC1, FPGS, and REG4 were measured by the Danenberg tumor profile (DTP) method. This study was performed in accordance with the ethics guidelines for clinical research with the approval of our institutional ethics committee of Osaka Red Cross Hospital, Japan. Written informed consent was obtained from all patients.
2.2.
DTP method
The DTP method consisted of laser microdissection of tumor cells from formalin-fixed, paraffin-embedded specimens, RNA extraction, and complementary DNA synthesis, with quantification of mRNA expression by real-time polymerase chain reaction (PCR). RNA was isolated from the formalin-fixed, paraffin-embedded specimens using a novel proprietary procedure (Response Genetics, Los Angeles, CA Patent Number 6,248,535), and complementary DNA sequences were amplified using quantitative PCR and a fluorescence-based realtime detection method (ABI PRISM 7900 Sequence Detection System [TaqMan]; Applied Biosystems, Foster City, CA). The PCR reaction mixture used contained primers, dATP, dCTP,
dGTP, and dUTP, MgCl2, and TaqMan buffer (all reagents were supplied by Applied Biosystems). The PCR conditions were 50 C for 10 s and 95 C for 10 min, followed by 42 cycles at 95 C for 15 s and 60 C for 1 min. The mRNA expression levels are expressed as values relative to those of ß-actin (ACTB) used as an internal reference [23e25].
2.3.
Statistical analysis
Categorical data were evaluated using Fisher exact test and continuous data by Wilcoxon rank-sum test. Disease-free survival (DFS) and overall survival (OS) were retrospectively assessed for each group of patients. OS was estimated from the date of diagnosis with liver metastases until death or last follow-up. DFS was defined as the time interval between hepatectomy and first postoperative recurrence or death. The probability of survival was calculated using the KaplaneMeier method, and differences between curves were compared using the log-rank test. Cox proportional hazards model was used to identify the independent prognostic factors of survival in all patients. Multivariate analysis was performed to examine association of the expression level of each gene with clinicopathologic factors and to determine factors independently related to gene expression. Two-sided P 0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS 19.0 software (SPSS, Chicago, IL).
2.4.
Cutoff and other definitions
The expression levels of each gene were categorized into low or high values with respect to the median: a high-expression
4
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 2 e The median expression of each marker in primary sites and liver metastatic sites. Site
Median Range P value
TS
DPD
Primary
Liver
Primary
Liver
Primary
Liver
0.115 0.0236e0.534 0.061
0.171 0.06738e1.301
0.00984 0.00965e0.0391 0.18
0.0170 0.0845e0.106
0.0618 0.0341e0.136 0.156
0.0563 0.0350e0.182
TP Median Range P value
0.154 0.00747e0.423 0.295
FPGS 0.178 0.0378e0.574
0.0230 0.0139e0.0579 0.375
group and a low-expression group. All patients were also divided into two groups according to the response to chemotherapy as follows: patients whose response was PR or CR were defined as a responder group, whereas those whose response was SD, PD, or NE were defined as a non-responder group. Furthermore, we defined the ratio of marker expression in the liver metastatic site to that in the primary site as the “L/P ratio”, to assess the difference of marker expression before and after chemotherapy indirectly.
3.
ERCC1
Results
The patients’ flow in this study is shown in Figure 1. All 43 patients received 5FU þ Oxaliplatin (L-OHP)-based chemotherapy and hepatic resection. For five of these markers except for REG4, seven pairs of these specimens were excluded from the analysis because a sufficient amount of mRNA could not be obtained. Thus, 36 paired samples were analyzed in this study. For REG4, only 25 samples in primary tumor and 16 in liver metastatic tumor were available for the analysis. The patient characteristics are shown in Table 1. Table 2 shows that association between the expression of each marker in primary sites and in liver metastatic sites. All markers seemed to show similar expression between primary and metastatic sites. The median follow-up was 42.6 mo. In terms of the DPD and TP collected from the primary tumor, the mean OS in the low-expression group was significantly higher than that in the high-expression group (67.4 mo versus 42.0 mo, P < 0.001 and 66.1 mo versus 49.1 mo, P ¼ 0.033, respectively; Figs. 2 and 3), but for DPD and TP collected from the liver metastatic site, there were no significant differences of OS between the two groups (53.7 mo versus 64.5 mo P ¼ 0.19 and 57.8 mo versus 58.5 mo P ¼ 0.87, respectively). The other four markers from both sites did not show any association of survival with the level of expression (Table 3). To explore the reason of the discrepancy of the same marker expressions from different sites, the association of the L/P ratio between the responder group and the non-responder group were assessed, using box plots (Fig. 4). The L/P ratios of DPD and TP were significantly higher in the responder group than in the non-responder group (P ¼ 0.034 and P ¼ 0.022, respectively). Univariate analysis of the expression of six markers from primary and liver metastatic sites revealed a significantly
REG4 0.0269 0.0132e0.0509
0.00450 0.000413e0.485 0.565
0.00840 0.000342e0.389
negative influence on the OS of high expression of DPD for the primary tumor and high expression of TP for the primary tumor. These two factors were also significant factors that negatively influenced DFS (Table 3). Multivariate analysis results are shown in Table 4. The analysis revealed a significantly negative influence of high expression of DPD.
4.
Discussion
Some groups have stated that mRNA expression levels in liver metastases and primary tumor from the same patients with colorectal cancer are similar or have some correlation [5,6]. Our results are consistent with these previous reports. Our results also showed the association of DPD and TP expression from primary tumor with OS, but no association from liver metastases. As far as we know, there have been no reports about the relationship of molecular biomarker expression collected from primary tumor and liver metastatic sites after chemotherapy with survival. There have also been few reports about assessing marker expression in liver metastatic sites and its association with prognosis [11,12]. One possible reason for this discrepancy might be that many molecular markers, such as TS, DPD, and TP, which are related to 5FU metabolism can be influenced by 5FU-combined chemotherapy. In our series, the L/P ratios of DPD and TP were also higher in the responder group than in the nonresponder group. The high level of TP in tumor tissue is essential for the efficacy of capecitabine and 50 -DFUR, associated with the metabolism of 5FU. The susceptibility of tumor tissues to 50 -DFUR has been reported to be enhanced by the transfection of TP [26]. Therefore, it is expected that TP upregulators would enhance the efficacy of capecitabine and 50 -DFUR. DPD are also said to be important enzymes determining the efficacy of 50 -DFUR [27]. These reports from in vitro studies indicate that TP and DPD may be upregulated in proportion to the response to 5FU chemotherapy. As mentioned previously, some reports about TP and DPD expression in primary tumors were inconsistent with our result, which may be partly because, in these reported studies, the adjuvant chemotherapy status of patients had not been well defined. Another possible reason for this discrepancy was that marker expression in primary colorectal cancer and in liver metastases may be originally different. If these markers were influenced by chemotherapy, the relationships between
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
5
Fig. 2 e OS according to DPD expression in primary site and liver metastatic site. (Color version of the figure is available online.)
marker expression in primary tumors and liver metastatic tumors could not be evaluated correctly in our study. If these markers were not influenced by chemotherapy, this hypothesis conflicted with our results that the expression of each marker in primary sites and in liver metastatic sites showed similar expression. In either case, we have to reevaluate the relation between these markers from two different sites in chemotherapy-naı¨ve setting. REG4 and FPGS, which are not directly involved in 5FU metabolism, showed no prognostic or predictive associations.
For REG4, reports have indicated that its overexpression is associated with a number of different cancers, including colorectal cancer [28], pancreatic cancer [20], gastric cancer [29], and prostate cancer [30]. However, the role of REG4 in carcinogenesis is not yet fully understood and few reported studies have investigated the relationship between its expression and the clinicopathologic features or prognostic outcomes in colorectal cancer. Numata et al. [31] stated that overexpression of REG4 was associated with a poor outcome. In our study, REG4 could be examined in only 25 cases of
Fig. 3 e OS according to TP expression in primary site and liver metastatic site. (Color version of the figure is available online.)
6
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 3 e Univariate analysis. Variables High versus low TS high DPD high TP high ERCC1 high EGPS high REG4 high
OS
DFS
Site
HR
95% CI
P value
HR
95% CI
P value
Primary Liver Primary Liver Primary Liver Primary Liver Primary Liver Primary Liver
1.2 2.81 9.53 0.42 3.68 0.9 1.66 0.73 0.67 0.9 180 65
0.24e5.98 0.81e9.78 2.41e37.6 0.11e1.59 1.02e14.7 0.27e2.98 0.50e5.56 0.22e2.41 0.20e2.30 0.27e0.96 0.18e17,000 0.14e55,000
0.82 0.11 0.001 0.2 0.048 0.87 0.41 0.61 0.52 0.86 0.14 0.47
0.6 2.22 6.98 0.48 4.06 1.27 1.12 0.79 0.59 1.17 1.95 1.44
0.13e2.80 0.65e7.63 1.78e27.4 0.13e1.82 1.07e15.4 0.39e4.17 0.34e3.69 0.23e2.52 0.173e2.03 0.55e2.50 0.75e5.10 0.44e4.76
0.51 0.21 0.005 0.28 0.039 0.69 0.85 0.66 0.4 0.69 0.17 0.55
CI ¼ confidence interval. P value 30 ng/mL at diagnosis
OS HR
95% CI
P value
7.83 1.45 1.3 0.43 1.51
1.59e38.51 0.30e6.87 0.16e10.40 0.05e3.67 0.25e9.10
0.011 0.64 0.8 0.44 0.65
CI ¼ confidence interval; CEA ¼ Carcinoembryonic antigen. P value
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Prognostic value of biomarkers in metastatic colorectal cancer patients Kozo Kataoka, MD,a,* Akiyoshi Kanazawa, MD, PhD, FACS,b Akio Nakajima, MD, PhD,a Ayane Yamaguchi, MD,a and Akira Arimoto, MD, PhDa a
Department of Surgery, Osaka Red-Cross Hospital, Osaka, Japan Department of Gastroenterological Surgery and Oncology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan
b
article info
abstract
Article history:
Backgrounds: The prognostic value of biomarkers in metastatic colorectal cancer (mCRC)
Received 29 July 2014
patients with liver metastases remains unclear. We assessed the difference of expression
Received in revised form
of biomarkers between primary tumors and liver metastases treated with chemotherapy in
9 September 2014
mCRC patients, as well as the prognostic value of these markers.
Accepted 3 October 2014
Methods: Forty-three mCRC patients with liver-limited disease from January 2007
Available online xxx
eNovember 2011 were analyzed. They all received resection of primary tumors followed by oxaliplatin-based chemotherapy. After chemotherapy, they all received hepatic resection.
Keywords:
Forty-three paired primary and metastatic tumor specimens were collected to measure the
Colorectal cancer
messenger RNA expression of six biomarkers by the Danenberg tumor profile method
Liver metastases
(thymidylate synthase, dihydropyrimidine dehydrogenase [DPD], excision repair cross-
Prognostic marker
complementing gene1, thymidine phosphorylase [TP], folylpolyglutamate synthase, and
TP
regenerating islet-derived family, member 4).
DPD
Results: Thirty-six patients’ messenger RNA was used for analysis. All markers showed similar expression between primary and metastatic sites. The low-expression group of Danenberg tumor profile and TP in the primary tumor showed significantly higher overall survival than the high-expression group (P < 0.001 and P ¼ 0.033), but for DPD and TP in liver metastases, there were no significant differences of overall survival between the two groups. The ratios of marker expression in liver metastatic site to that in primary site of DPD and TP were significantly higher in chemo-responders than in non-chemo-responders (P ¼ 0.034 and P ¼ 0.022). Conclusions: Biomarkers’ expressions in liver metastases were similar to those in the primary tumor. DPD and TP in the primary lesion may be a prognostic factor in chemotherapy-naı¨ve mCRC patients with liver-limited disease, but those in liver tumor were not. Further validated analysis to our results would be warranted. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Osaka Red-Cross Hospital, 5-30, Fudegasaki, Tennoji, Osaka 543 8555, Japan. Tel.: þ81 6 6774 5111; fax: þ81 6 6774 5131. E-mail address: [email protected] (K. Kataoka). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.006
2
1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Introduction
Colorectal cancer is one of the most common causes of cancer-related mortality worldwide, and the liver is the most common, and often the only, metastatic site [1,2]. Surgical resection of colorectal liver metastases (CLM) is considered the only curative therapy, but most metastatic colorectal cancer (mCRC) patients with CLM have unresectable disease [3,4]. It is important to develop the strategy of chemotherapy based on the expression of biomarkers to improve the survival of CLM patients. One of the concerns about biomarkers for CLM patients is to reveal the differences of marker expression between primary lesion and liver metastatic lesion. Some reports have described that the messenger RNA (mRNA) expression levels of several biomarkers (thymidylate synthase [TS], dihydropyrimidine dehydrogenase [DPD], thymidine phosphorylase [TP], and others) in the primary site and the liver metastatic site were similar or showed a positive correlation if laser-captured microdissection was used [5,6]. However, the association of expression of these markers between primary tumor site and metastatic site is not fully understood. In this study, we selected six specific genes as follows: TS, DPD, TP, excision repair cross-complementing gene 1 (ERCC1), folylpolyglutamate synthase (FPGS), and regenerating isletderived family, member 4 (REG4). TS, DPD, and TP are involved in the metabolism of fluoropyrimidines, and there are many reports about these markers in colorectal cancer [7e17]. ERCC1 is an excision nuclease within the nucleotide excision repair pathway that plays a major role in repairing platinum-induced DNA adducts [18]. There are some reports that ERCC1 might
predict the efficacy of oxaliplatin-combined chemotherapy and that expression of ERCC1 was associated with prognosis of colorectal cancer, but their predictive and prognostic value is still unclear [11,16,19]. Folate-metabolizing enzyme FPGS is involved in the metabolism of folic acid [14] and REG4, which is a member of the REG family that acts as an antiapoptotic factor through the AKT signaling pathway [20,21]. Using these biomarkers collected from the same patients’ primary tumor tissues and liver metastatic tissues, we assessed the difference of their expression between primary site and liver metastatic site treated with chemotherapy in CLM patients retrospectively. We also assessed the prognostic value of markers collected from two different sites of the same patient.
2.
Methods
2.1.
Patients
Forty-three mCRC patients with resectable liver-limited disease from January 2007eNovember 2011 were assessed. mCRC patients who had extrahepatic disease and who had unresectable liver metastases were excluded from the analysis. Resectability was decided based on the size of the remnant liver volume (>30%) and expected function after the removal of all metastases, regardless of the number and size of the liver metastases. If metastases infiltrated (1) all hepatic veins, (2) both hepatic arteries and 3 both portal vein branches, these patients were defined as unresectable. Patients who received chemotherapy within 12 mo of diagnosis of mCRC with liver-limited disease were also excluded from the analysis.
Fig. 1 e Patients’ flow. LLD, liver-limited disease. (Color version of the figure is available online.)
3
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 1 e Patients’ characteristics. 2007.1e2011.11
n ¼ 36
Median (range) Men Women T1, 2 T3, 4 N0 N (þ) Rectum Colon Synchronous Metachronous Median (range) Median (range) mFOLFOX6/Xelox mFOLFOX6/Xelox þ bevacizumab mFOLFOX6/Xelox þ cetuximab Median (range) Yes No CR, PR SD PD NE Median (range) 0e1 2
66 (27e81) 23 13 3 33 11 25 11 25 15 21 2 (1e11) 2.6 (0.9e12) 27 6 3 6 (3e22) 28 8 13 14 7 2 9.8 (1.5e1215) 36 0
Variables Age (y) Sex T category of the primary cancer Lymphatic spread of the primary cancer Location of primary tumor Interval to metastasis Number of liver metastases Size of largest liver metastasis (cm) Preoperative chemotherapy
Cycles of chemotherapy Adjuvant chemotherapy Clinical response to chemotherapy
Serum CEA level (ng/mL) ECOG performance status
CR ¼ Complete Response; PR ¼ Partial Response; SD ¼ Stable Disease; PD ¼ Progressive Disease; NE ¼ Not all Evaluated; CEA ¼ Carcinoembryonic antigen; ECOG ¼ Eastern Cooperative Oncology Group.
First, they underwent colorectomy if metastasis was synchronous. After that, they received oxaliplatin-based chemotherapy. The response to chemotherapy was evaluated using Response Evaluation Criteria in Solid Tumors criteria (version 1.1) [22]. After chemotherapy, they received hepatic resection. The interval between the last administration of chemotherapy and resection was generally about 28 d, and 42 d especially when bevacizumab was used. Tumor tissues were collected from both primary tumors and liver metastatic tumors. Paired mRNA expression levels of TS, DPD, TP, ERCC1, FPGS, and REG4 were measured by the Danenberg tumor profile (DTP) method. This study was performed in accordance with the ethics guidelines for clinical research with the approval of our institutional ethics committee of Osaka Red Cross Hospital, Japan. Written informed consent was obtained from all patients.
2.2.
DTP method
The DTP method consisted of laser microdissection of tumor cells from formalin-fixed, paraffin-embedded specimens, RNA extraction, and complementary DNA synthesis, with quantification of mRNA expression by real-time polymerase chain reaction (PCR). RNA was isolated from the formalin-fixed, paraffin-embedded specimens using a novel proprietary procedure (Response Genetics, Los Angeles, CA Patent Number 6,248,535), and complementary DNA sequences were amplified using quantitative PCR and a fluorescence-based realtime detection method (ABI PRISM 7900 Sequence Detection System [TaqMan]; Applied Biosystems, Foster City, CA). The PCR reaction mixture used contained primers, dATP, dCTP,
dGTP, and dUTP, MgCl2, and TaqMan buffer (all reagents were supplied by Applied Biosystems). The PCR conditions were 50 C for 10 s and 95 C for 10 min, followed by 42 cycles at 95 C for 15 s and 60 C for 1 min. The mRNA expression levels are expressed as values relative to those of ß-actin (ACTB) used as an internal reference [23e25].
2.3.
Statistical analysis
Categorical data were evaluated using Fisher exact test and continuous data by Wilcoxon rank-sum test. Disease-free survival (DFS) and overall survival (OS) were retrospectively assessed for each group of patients. OS was estimated from the date of diagnosis with liver metastases until death or last follow-up. DFS was defined as the time interval between hepatectomy and first postoperative recurrence or death. The probability of survival was calculated using the KaplaneMeier method, and differences between curves were compared using the log-rank test. Cox proportional hazards model was used to identify the independent prognostic factors of survival in all patients. Multivariate analysis was performed to examine association of the expression level of each gene with clinicopathologic factors and to determine factors independently related to gene expression. Two-sided P 0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS 19.0 software (SPSS, Chicago, IL).
2.4.
Cutoff and other definitions
The expression levels of each gene were categorized into low or high values with respect to the median: a high-expression
4
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 2 e The median expression of each marker in primary sites and liver metastatic sites. Site
Median Range P value
TS
DPD
Primary
Liver
Primary
Liver
Primary
Liver
0.115 0.0236e0.534 0.061
0.171 0.06738e1.301
0.00984 0.00965e0.0391 0.18
0.0170 0.0845e0.106
0.0618 0.0341e0.136 0.156
0.0563 0.0350e0.182
TP Median Range P value
0.154 0.00747e0.423 0.295
FPGS 0.178 0.0378e0.574
0.0230 0.0139e0.0579 0.375
group and a low-expression group. All patients were also divided into two groups according to the response to chemotherapy as follows: patients whose response was PR or CR were defined as a responder group, whereas those whose response was SD, PD, or NE were defined as a non-responder group. Furthermore, we defined the ratio of marker expression in the liver metastatic site to that in the primary site as the “L/P ratio”, to assess the difference of marker expression before and after chemotherapy indirectly.
3.
ERCC1
Results
The patients’ flow in this study is shown in Figure 1. All 43 patients received 5FU þ Oxaliplatin (L-OHP)-based chemotherapy and hepatic resection. For five of these markers except for REG4, seven pairs of these specimens were excluded from the analysis because a sufficient amount of mRNA could not be obtained. Thus, 36 paired samples were analyzed in this study. For REG4, only 25 samples in primary tumor and 16 in liver metastatic tumor were available for the analysis. The patient characteristics are shown in Table 1. Table 2 shows that association between the expression of each marker in primary sites and in liver metastatic sites. All markers seemed to show similar expression between primary and metastatic sites. The median follow-up was 42.6 mo. In terms of the DPD and TP collected from the primary tumor, the mean OS in the low-expression group was significantly higher than that in the high-expression group (67.4 mo versus 42.0 mo, P < 0.001 and 66.1 mo versus 49.1 mo, P ¼ 0.033, respectively; Figs. 2 and 3), but for DPD and TP collected from the liver metastatic site, there were no significant differences of OS between the two groups (53.7 mo versus 64.5 mo P ¼ 0.19 and 57.8 mo versus 58.5 mo P ¼ 0.87, respectively). The other four markers from both sites did not show any association of survival with the level of expression (Table 3). To explore the reason of the discrepancy of the same marker expressions from different sites, the association of the L/P ratio between the responder group and the non-responder group were assessed, using box plots (Fig. 4). The L/P ratios of DPD and TP were significantly higher in the responder group than in the non-responder group (P ¼ 0.034 and P ¼ 0.022, respectively). Univariate analysis of the expression of six markers from primary and liver metastatic sites revealed a significantly
REG4 0.0269 0.0132e0.0509
0.00450 0.000413e0.485 0.565
0.00840 0.000342e0.389
negative influence on the OS of high expression of DPD for the primary tumor and high expression of TP for the primary tumor. These two factors were also significant factors that negatively influenced DFS (Table 3). Multivariate analysis results are shown in Table 4. The analysis revealed a significantly negative influence of high expression of DPD.
4.
Discussion
Some groups have stated that mRNA expression levels in liver metastases and primary tumor from the same patients with colorectal cancer are similar or have some correlation [5,6]. Our results are consistent with these previous reports. Our results also showed the association of DPD and TP expression from primary tumor with OS, but no association from liver metastases. As far as we know, there have been no reports about the relationship of molecular biomarker expression collected from primary tumor and liver metastatic sites after chemotherapy with survival. There have also been few reports about assessing marker expression in liver metastatic sites and its association with prognosis [11,12]. One possible reason for this discrepancy might be that many molecular markers, such as TS, DPD, and TP, which are related to 5FU metabolism can be influenced by 5FU-combined chemotherapy. In our series, the L/P ratios of DPD and TP were also higher in the responder group than in the nonresponder group. The high level of TP in tumor tissue is essential for the efficacy of capecitabine and 50 -DFUR, associated with the metabolism of 5FU. The susceptibility of tumor tissues to 50 -DFUR has been reported to be enhanced by the transfection of TP [26]. Therefore, it is expected that TP upregulators would enhance the efficacy of capecitabine and 50 -DFUR. DPD are also said to be important enzymes determining the efficacy of 50 -DFUR [27]. These reports from in vitro studies indicate that TP and DPD may be upregulated in proportion to the response to 5FU chemotherapy. As mentioned previously, some reports about TP and DPD expression in primary tumors were inconsistent with our result, which may be partly because, in these reported studies, the adjuvant chemotherapy status of patients had not been well defined. Another possible reason for this discrepancy was that marker expression in primary colorectal cancer and in liver metastases may be originally different. If these markers were influenced by chemotherapy, the relationships between
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
5
Fig. 2 e OS according to DPD expression in primary site and liver metastatic site. (Color version of the figure is available online.)
marker expression in primary tumors and liver metastatic tumors could not be evaluated correctly in our study. If these markers were not influenced by chemotherapy, this hypothesis conflicted with our results that the expression of each marker in primary sites and in liver metastatic sites showed similar expression. In either case, we have to reevaluate the relation between these markers from two different sites in chemotherapy-naı¨ve setting. REG4 and FPGS, which are not directly involved in 5FU metabolism, showed no prognostic or predictive associations.
For REG4, reports have indicated that its overexpression is associated with a number of different cancers, including colorectal cancer [28], pancreatic cancer [20], gastric cancer [29], and prostate cancer [30]. However, the role of REG4 in carcinogenesis is not yet fully understood and few reported studies have investigated the relationship between its expression and the clinicopathologic features or prognostic outcomes in colorectal cancer. Numata et al. [31] stated that overexpression of REG4 was associated with a poor outcome. In our study, REG4 could be examined in only 25 cases of
Fig. 3 e OS according to TP expression in primary site and liver metastatic site. (Color version of the figure is available online.)
6
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e8
Table 3 e Univariate analysis. Variables High versus low TS high DPD high TP high ERCC1 high EGPS high REG4 high
OS
DFS
Site
HR
95% CI
P value
HR
95% CI
P value
Primary Liver Primary Liver Primary Liver Primary Liver Primary Liver Primary Liver
1.2 2.81 9.53 0.42 3.68 0.9 1.66 0.73 0.67 0.9 180 65
0.24e5.98 0.81e9.78 2.41e37.6 0.11e1.59 1.02e14.7 0.27e2.98 0.50e5.56 0.22e2.41 0.20e2.30 0.27e0.96 0.18e17,000 0.14e55,000
0.82 0.11 0.001 0.2 0.048 0.87 0.41 0.61 0.52 0.86 0.14 0.47
0.6 2.22 6.98 0.48 4.06 1.27 1.12 0.79 0.59 1.17 1.95 1.44
0.13e2.80 0.65e7.63 1.78e27.4 0.13e1.82 1.07e15.4 0.39e4.17 0.34e3.69 0.23e2.52 0.173e2.03 0.55e2.50 0.75e5.10 0.44e4.76
0.51 0.21 0.005 0.28 0.039 0.69 0.85 0.66 0.4 0.69 0.17 0.55
CI ¼ confidence interval. P value 30 ng/mL at diagnosis
OS HR
95% CI
P value
7.83 1.45 1.3 0.43 1.51
1.59e38.51 0.30e6.87 0.16e10.40 0.05e3.67 0.25e9.10
0.011 0.64 0.8 0.44 0.65
CI ¼ confidence interval; CEA ¼ Carcinoembryonic antigen. P value
