- Email: [email protected]
Predictive biomarkers for the efficacy of concurrent chemoradiotherapy for patients with colorectal cancer
Biomarkers and Genomic Medicine (2014) 6, 163e166
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
SHORT COMMUNICATION
Predictive biomarkers for the efficacy of concurrent chemoradiotherapy for patients with colorectal cancer Hsin-Ju Wu a, Hsueh-Chiao Liu a,b, Yu-Tang Chang c, Shiu-Ru Lin d,*, Long-Sen Chang a,** a
Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan Division of Laboratory Medicine, Fooying University Hospital, Pingtung, Taiwan c Division of Pediatric Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan d Division of Medical Research, Fooying University Hospital, Pingtung, Taiwan b
Received 1 July 2014; received in revised form 23 August 2014; accepted 25 August 2014
Available online 1 October 2014
KEYWORDS colorectal cancer; concurrent chemoradiotherapy response-related genes; efficacy of concurrent chemoradiotherapy; gene chip; molecular markers
Abstract Colorectal cancer is a common gastrointestinal malignancy. Radiation combined with chemotherapy (also known as concurrent chemoradiotherapy or CCRT) is often used prior to surgery for treating severe cases of colorectal cancer. However, responses of individual tumors to CCRT differ. Therefore, in light of the variability in radiation sensitivity among different tumors, identifying the factors that can be applied to predict CCRT efficacy prior to treatment will aid in making decisions regarding an appropriate treatment strategy. In the present study, we used a gene chip to analyze the expression of candidate genes in the tumor cells of colorectal cancer patients prior to and after treatment with CCRT, in order to identify molecular markers that can predict the efficacy of CCRT. First, we selected a total of 15 CCRT candidate genes based on the results of previous studies, which used the microarray method to select CCRT response-related genes that were also related to tumor malignancy. We collected preoperative CCRT tumor tissues from 17 colorectal cancer patients for whom the efficacy of CCRT had already been determined and used gene chips to analyze the expression of CCRT-related genes in the tissues of these patients. We then compared the results for the expression of CCRT-related genes with those for the clinical efficacy of CCRT. Of the 15 candidate genes, five genes (CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2) were upregulated in >29.4% and one gene (MET ) was downregulated in 23.5% of the total patients after
* Corresponding author. Division of Medical Research, Fooyin University Hospital, Number 5, Jhongshan Road, Donggang Township, Pingtung 928, Taiwan. ** Corresponding author. Institute of Biomedical Sciences, National Sun Yat-Sen University, 70 Lienhai Road, Kaohsiung 804, Taiwan. E-mail addresses: [email protected] (S.-R. Lin), [email protected] (L.-S. Chang). http://dx.doi.org/10.1016/j.bgm.2014.08.007 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
164
H.-J. Wu et al. treatment with CCRT, indicating that these genes may be potential predictive markers for CCRT efficacy. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. Most cancer treatments are primarily based on surgical resection, but surgery is not suitable for treating all types of cancers (for example, in cases where the tumor has spread) due to some limitations. As such, radiation therapy or chemotherapy is often used as an adjuvant therapy. Effectiveness of radiotherapy treatment is not limited by the lesion location. Preoperative radiation therapy usually yields better results by shrinking the tumor, thus increasing the chances of complete surgical resection of the tumor. Therefore, a combination of comprehensive surgery and radiotherapy can provide local control to improve treatment efficacy.1e3 According to the 2010 United States National Comprehensive Cancer Network Version 1 guidelines, radiation prior to surgery combined with chemotherapy (CCRT) is the recommended treatment option for locally advanced CRCs (T3e4 or N1e2). Studies have reported that the role of chemical treatment drugs (such as 5-fluorouracil) in preoperative CCRT for patients with advanced or unresectable CRCs is to enhance the effectiveness of radiation therapy in killing cancer cells. In other words, radiation therapy provides the major therapeutic effects of CCRT, whereas chemotherapy supplements those therapeutic effects.4e6 Clinically, approximately 30e40% of patients are unable to obtain the benefit of a decreased tumor stage (downstaging) after receiving CCRT. In patients for whom CCRT show poor efficacy, preoperative CCRT is more harmful than helpful because it not only delays the timing of tumor resection, but also results in immunosuppression, causes patients to suffer unnecessarily, and is a waste of medical resources. As such, if we can predict the efficacy of CCRT, we can use medical resources and treat the disease more effectively. Such predictive ability would thus not only reduce the chances of recurrence, but also greatly enhance the likelihood of organ retention, particularly the affected organs. The reason for individual differences in sensitivity to CCRT is that different tumors exhibit different expression profiles of related genes. Because cellular responses rely, in part, on changes in gene expression, the extent to which the radiation-responsive genes are induced or repressed influences how the cells deal with radiation exposure, where individual variability in radiation sensitivity is observed at the gene expression level.7e9 Therefore, in the present study, we sought to determine the molecular markers that can predict the efficacy of CCRT, and then explored the impact of each genetic marker using a chip platform that was established by Wang et al.10 This platform possesses the various advantages of biochips, has a lower cost, and exhibits
better usability, higher reproducibility, and higher accuracy compared to traditional chips. Results obtained with this technology are consistent with the real-time quantitative polymerase chain reaction test results. Therefore, it is possible to predict the efficiency of clinical treatment by establishing a chip system; these prediction results may serve as important references and help clinical doctors in treatment planning prior to initiation of therapy.
Materials and methods Collection of specimen In our study, data were gathered for 17 patients for whom the clinical efficacy of CCRT was already known, excluding any tissue specimens from CRC patients who also had other cancers at the same time. For each patient, a biopsy sample of the tumor tissue was taken prior to and after treatment with CCRT prior to surgery, and kept on ice or in liquid nitrogen. All the patients received preoperative radiotherapy (45e50 Gy), and all were also treated with chemotherapy during the radiotherapy process. All the patients then subsequently underwent surgery. Data on the clinical characteristics of each patient, including patient age, gender, tumor size, location, histological type, clinical stage, metastasis situation, and other information, were collected.
Selection of genes and oligonucleotide design Fifteen candidate genes were selected based on the microarray research of our previous studies and other recent studies11,12 in which the microarray method was used to compare the expressions of candidate genes in radiosensitive and radiation-resistant cells, as well as to determine which genes are related to tumor malignancy. Using the Oligo Explorer software program (Gene Link, Inc., New York, NY, USA), we designed a detection chip to identify the nucleic acid sequences of candidate genes related to radiotherapy effects.
Preparation of gene chip The synthetic oligonucleotides were dissolved at a concentration of 10 nM in double distilled water. We then used BioDOT AD1500 (BioDot Inc., Irvine, CA, USA) to automatically dot candidate genes, B-actin (positive control), and dimethyl sulfoxide (blank) on nylon membrane in triplicate. After quick drying and adhesive fixing, the prototype chip was obtained.
Predictive biomarkers for CCRT efficacy in CRC patients
Gene validation using gene chip A GeneCling Enzymatic Gene Chip Detection Kit (CaryGene Biotechnology Co., Ltd, Kaohsiung, Taiwan) was used for carrying out the following steps. First, beads were added for RNA extraction, and an RT buffer and an RT enzyme were then added to synthesize cDNA. Biotin Mix Label was then added to cDNA for the synthesis of biotin-labeled probes (probe synthesis). Then the labeled cDNA was hybridized with the prepared chip at 42 C for 6e17 hours. A wash buffer was then used to wash away the probes that were not hybridized, followed by the addition of streptavidin-AP and nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP) mixture and incubation at 37 C to conduct a color reaction. After coloration the chips were completely dried; the colorimetric values of the individual genes were analyzed using software, and these values were then translated so as to indicate the relative intensities of the various gene expressions. If the gene presented a color density of greater than two-fold compared to the positive control (beta-actin), it was defined as positive. If the gene expression was less than two-fold compared to the positive control, it was classed as negative.
Results
165 Table 1 Clinicopathological features of 17 colorectal cancer patients. Characteristics Sex Male Female Age (y) <60 >60 Tumor size (cm) >5 <5 Stage I þ II III þ IV Vascular invasion Negative Positive Perineural invasion Negative Positive Liver metastasis Yes No
Number of cases (n Z 17)
%
11 6
64 36
10 7
59 41
9 8
53 47
5 12
29 71
8 9
47 53
13 4
77 23
3 14
18 82
Clinicopathological features of 17 CRC patients Clinicopathological features of 17 CRC patients [11 men (64%) and six women (36%)] were recorded. The average age was 54.7 years (range: 30e72 years). Table 1 shows the clinicopathological characteristics of these 17 patients, all of whom met the stage IeIII CRC criteria of the Union for International Cancer Control.
Expression of 15 candidate genes in CRC patients after treatment with CCRT We compared the variations of gene expressions in the tissues of the 17 patients prior to and after treatment with CCRT. If the expression of a candidate gene was positive prior to treatment and became negative after treatment with CCRT, we defined the gene as being downregulated after treatment. By contrast, if the expression of a candidate gene was negative prior to treatment and became positive after treatment with CCRT, we defined the gene as being upregulated after treatment. Of the 15 candidate genes, CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2 genes were differentially downregulated in, respectively, 52.9%, 52.9%, 52.9%, 29.4%, and 29.4% of the total patients after treatment with CCRT. One gene, MET, was upregulated in 23.5% of the total patients after treatment with CCRT (Table 2). Although OLFM4 and hTER genes showed a higher proportion in downregulated cases after treatment with CCRT, they did not show differential proportion in upregulated cases.
Discussion Preoperative radiotherapy has widely been used to improve local control of disease and survival of patients following
treatment of rectal cancer. There is great variability, however, in the CCRT responses of different individuals due to the lack of knowledge of how radiation-responsive genes are induced or repressed and how cells deal with radiation exposure.9 Therefore, we developed markers for predicting the effectiveness of CCRT in CRC patients. In the present study, we demonstrated the overexpression patterns in CRC patients prior to and after
Table 2 Gene expression of 15 target genes using gene chip in 17 colorectal cancer patients treated with CCRT. Gene
Downregulated cases after CCRT (N Z 17)
Upregulated cases after CCRT (N Z 17)
CK-20 ELAVL4 EV12B TM4SF3 ATPA2 OLFM4 hTERT CD55 S100B KCTD2 CEA KLK7 CK-19 MUC1 MET
9 9 9 5 5 9 7 4 3 3 2 2 1 1 0
0 2 2 0 1 4 4 4 0 1 2 2 2 2 4
(52.9) (52.9) (52.9) (29.4) (29.4) (52.9) (41.1) (23.5) (17.6) (17.6) (11.7) (11.7) (5.8) (5.8) (0)
Data are presented as n (%). CCRT Z concurrent chemoradiotherapy.
(0) (11.7) (11.7) (0) (5.8) (23.5) (23.5) (23.5) (0) (5.8) (11.7) (11.7) (11.7) (11.7) (23.5)
166 treatment with CCRT. Of the 15 CRC-related genes, five genes (CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2) had a downregulation rate of >29.4% of the total patients after treatment with CCRT. Additionally, one gene (MET ) had an upregulation rate of >20% of the total patients after treatment with CCRT. These differentially expressed genes show potential to be predictive markers for CCRT efficacy. Both upregulation of CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2 genes that can be detected in the tumor of patients prior to CCRT and upregulation of MET gene that can be detected in the tumor after treatment with low-dose radiation can indicate the radiosensitivity. Through either kind of detection methods, it can provide the predictive results of treatment efficacy for the clinician prior to applying the CCRT to patients. However, because of the small sample size analyzed in this study, there was no statistically significant correlation between the expression of the 15 genes and the clinicopathological features of the CRC patients. In addition, upand downregulation rates of the 15 genes in the CRC patients after treatment with CCRT were not very high. Higher patient numbers would help increase the sensitivity in detecting any differences, if they exist. Therefore, further investigation should be conducted with a large number of samples to verify any potential clinical value. It is hoped that the study results can help clinicians plan an effective course of treatment.
H.-J. Wu et al.
3.
4.
5.
6.
7.
8.
9. 10.
Conflicts of interest The authors declare no conflicts of interest. 11.
References 1. Swedish Rectal Cancer Trial. Improved survival with preoperative radiotherapy in resectable rectal cancer. N Engl J Med. 1997;336:980e987. 2. Gerard A, Buyse M, Nordlinger B, et al. Preoperative radiotherapy as adjuvant treatment in rectal cancer: final results of
12.
a randomized study of the European Organization for Research and Treatment of Cancer (EORTC). Ann Surg. 1988;208: 606e614. Goldberg PA, Nicholls RJ, Porter NH, et al. Long-term results of a randomised trial of short-course low-dose adjuvant preoperative radiotherapy for rectal cancer: reduction in local treatment failure. Eur J Cancer. 1994;30:1602e1606. Frykholm GJ, Pahlman L, Glimelius B. Combined chemo- and radiotherapy vs. radiotherapy alone in the treatment of primary, nonresectable adenocarcinoma of the rectum. Int J Radiat Oncol Biol Phys. 2001;50:427e434. Bosset JF, Calais G, Mineur L, et al. Enhanced tumorocidal effect of chemotherapy with preoperative radiotherapy for rectal cancer: preliminary resultsdEORTC 22921. J Clin Oncol. 2005;23:5620e5627. Hendlisz A, Van den Eynde M, Peeters M, et al. Phase III trial comparing protracted intravenous fluorouracil infusion alone or with yttrium-90 resin microspheres radioembolization for liver-limited metastatic colorectal cancer. J Clin Oncol. 2010; 28:3687e3694. Huang MY, Wang JY, Chang HJ, et al. CDC25A, VAV1, TP73, BRCA1 and ZAP70 gene overexpression correlates with radiation response in colorectal cancer. Oncol Rep. 2011;25: 1297e1306. Amundson SA, Do KT, Vinikoor LC, et al. Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Cancer Res. 2008;68:415e424. Correa CR, Cheung VG. Genetic variation in radiation-induced expression phenotypes. Am J Hum Genet. 2004;75:885e890. Wang JY, Yeh CS, Chen YF, et al. Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer. Int J Mol Med. 2006;17: 737e747. Huang MY, Wang HM, Chang HJ, et al. Overexpression of S100B, TM4SF4, and OLFM4 genes is correlated with liver metastasis in Taiwanese colorectal cancer patients. DNA Cell Biol. 2012;3: 43e49. Huang MY, Wang HM, Tok TS, et al. EVI2B, ATP2A2, S100B, TM4SF3, and OLFM4 as potential prognostic markers for postoperative Taiwanese colorectal cancer patients. DNA Cell Biol. 2012;31:625e635.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
SHORT COMMUNICATION
Predictive biomarkers for the efficacy of concurrent chemoradiotherapy for patients with colorectal cancer Hsin-Ju Wu a, Hsueh-Chiao Liu a,b, Yu-Tang Chang c, Shiu-Ru Lin d,*, Long-Sen Chang a,** a
Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan Division of Laboratory Medicine, Fooying University Hospital, Pingtung, Taiwan c Division of Pediatric Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan d Division of Medical Research, Fooying University Hospital, Pingtung, Taiwan b
Received 1 July 2014; received in revised form 23 August 2014; accepted 25 August 2014
Available online 1 October 2014
KEYWORDS colorectal cancer; concurrent chemoradiotherapy response-related genes; efficacy of concurrent chemoradiotherapy; gene chip; molecular markers
Abstract Colorectal cancer is a common gastrointestinal malignancy. Radiation combined with chemotherapy (also known as concurrent chemoradiotherapy or CCRT) is often used prior to surgery for treating severe cases of colorectal cancer. However, responses of individual tumors to CCRT differ. Therefore, in light of the variability in radiation sensitivity among different tumors, identifying the factors that can be applied to predict CCRT efficacy prior to treatment will aid in making decisions regarding an appropriate treatment strategy. In the present study, we used a gene chip to analyze the expression of candidate genes in the tumor cells of colorectal cancer patients prior to and after treatment with CCRT, in order to identify molecular markers that can predict the efficacy of CCRT. First, we selected a total of 15 CCRT candidate genes based on the results of previous studies, which used the microarray method to select CCRT response-related genes that were also related to tumor malignancy. We collected preoperative CCRT tumor tissues from 17 colorectal cancer patients for whom the efficacy of CCRT had already been determined and used gene chips to analyze the expression of CCRT-related genes in the tissues of these patients. We then compared the results for the expression of CCRT-related genes with those for the clinical efficacy of CCRT. Of the 15 candidate genes, five genes (CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2) were upregulated in >29.4% and one gene (MET ) was downregulated in 23.5% of the total patients after
* Corresponding author. Division of Medical Research, Fooyin University Hospital, Number 5, Jhongshan Road, Donggang Township, Pingtung 928, Taiwan. ** Corresponding author. Institute of Biomedical Sciences, National Sun Yat-Sen University, 70 Lienhai Road, Kaohsiung 804, Taiwan. E-mail addresses: [email protected] (S.-R. Lin), [email protected] (L.-S. Chang). http://dx.doi.org/10.1016/j.bgm.2014.08.007 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
164
H.-J. Wu et al. treatment with CCRT, indicating that these genes may be potential predictive markers for CCRT efficacy. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. Most cancer treatments are primarily based on surgical resection, but surgery is not suitable for treating all types of cancers (for example, in cases where the tumor has spread) due to some limitations. As such, radiation therapy or chemotherapy is often used as an adjuvant therapy. Effectiveness of radiotherapy treatment is not limited by the lesion location. Preoperative radiation therapy usually yields better results by shrinking the tumor, thus increasing the chances of complete surgical resection of the tumor. Therefore, a combination of comprehensive surgery and radiotherapy can provide local control to improve treatment efficacy.1e3 According to the 2010 United States National Comprehensive Cancer Network Version 1 guidelines, radiation prior to surgery combined with chemotherapy (CCRT) is the recommended treatment option for locally advanced CRCs (T3e4 or N1e2). Studies have reported that the role of chemical treatment drugs (such as 5-fluorouracil) in preoperative CCRT for patients with advanced or unresectable CRCs is to enhance the effectiveness of radiation therapy in killing cancer cells. In other words, radiation therapy provides the major therapeutic effects of CCRT, whereas chemotherapy supplements those therapeutic effects.4e6 Clinically, approximately 30e40% of patients are unable to obtain the benefit of a decreased tumor stage (downstaging) after receiving CCRT. In patients for whom CCRT show poor efficacy, preoperative CCRT is more harmful than helpful because it not only delays the timing of tumor resection, but also results in immunosuppression, causes patients to suffer unnecessarily, and is a waste of medical resources. As such, if we can predict the efficacy of CCRT, we can use medical resources and treat the disease more effectively. Such predictive ability would thus not only reduce the chances of recurrence, but also greatly enhance the likelihood of organ retention, particularly the affected organs. The reason for individual differences in sensitivity to CCRT is that different tumors exhibit different expression profiles of related genes. Because cellular responses rely, in part, on changes in gene expression, the extent to which the radiation-responsive genes are induced or repressed influences how the cells deal with radiation exposure, where individual variability in radiation sensitivity is observed at the gene expression level.7e9 Therefore, in the present study, we sought to determine the molecular markers that can predict the efficacy of CCRT, and then explored the impact of each genetic marker using a chip platform that was established by Wang et al.10 This platform possesses the various advantages of biochips, has a lower cost, and exhibits
better usability, higher reproducibility, and higher accuracy compared to traditional chips. Results obtained with this technology are consistent with the real-time quantitative polymerase chain reaction test results. Therefore, it is possible to predict the efficiency of clinical treatment by establishing a chip system; these prediction results may serve as important references and help clinical doctors in treatment planning prior to initiation of therapy.
Materials and methods Collection of specimen In our study, data were gathered for 17 patients for whom the clinical efficacy of CCRT was already known, excluding any tissue specimens from CRC patients who also had other cancers at the same time. For each patient, a biopsy sample of the tumor tissue was taken prior to and after treatment with CCRT prior to surgery, and kept on ice or in liquid nitrogen. All the patients received preoperative radiotherapy (45e50 Gy), and all were also treated with chemotherapy during the radiotherapy process. All the patients then subsequently underwent surgery. Data on the clinical characteristics of each patient, including patient age, gender, tumor size, location, histological type, clinical stage, metastasis situation, and other information, were collected.
Selection of genes and oligonucleotide design Fifteen candidate genes were selected based on the microarray research of our previous studies and other recent studies11,12 in which the microarray method was used to compare the expressions of candidate genes in radiosensitive and radiation-resistant cells, as well as to determine which genes are related to tumor malignancy. Using the Oligo Explorer software program (Gene Link, Inc., New York, NY, USA), we designed a detection chip to identify the nucleic acid sequences of candidate genes related to radiotherapy effects.
Preparation of gene chip The synthetic oligonucleotides were dissolved at a concentration of 10 nM in double distilled water. We then used BioDOT AD1500 (BioDot Inc., Irvine, CA, USA) to automatically dot candidate genes, B-actin (positive control), and dimethyl sulfoxide (blank) on nylon membrane in triplicate. After quick drying and adhesive fixing, the prototype chip was obtained.
Predictive biomarkers for CCRT efficacy in CRC patients
Gene validation using gene chip A GeneCling Enzymatic Gene Chip Detection Kit (CaryGene Biotechnology Co., Ltd, Kaohsiung, Taiwan) was used for carrying out the following steps. First, beads were added for RNA extraction, and an RT buffer and an RT enzyme were then added to synthesize cDNA. Biotin Mix Label was then added to cDNA for the synthesis of biotin-labeled probes (probe synthesis). Then the labeled cDNA was hybridized with the prepared chip at 42 C for 6e17 hours. A wash buffer was then used to wash away the probes that were not hybridized, followed by the addition of streptavidin-AP and nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP) mixture and incubation at 37 C to conduct a color reaction. After coloration the chips were completely dried; the colorimetric values of the individual genes were analyzed using software, and these values were then translated so as to indicate the relative intensities of the various gene expressions. If the gene presented a color density of greater than two-fold compared to the positive control (beta-actin), it was defined as positive. If the gene expression was less than two-fold compared to the positive control, it was classed as negative.
Results
165 Table 1 Clinicopathological features of 17 colorectal cancer patients. Characteristics Sex Male Female Age (y) <60 >60 Tumor size (cm) >5 <5 Stage I þ II III þ IV Vascular invasion Negative Positive Perineural invasion Negative Positive Liver metastasis Yes No
Number of cases (n Z 17)
%
11 6
64 36
10 7
59 41
9 8
53 47
5 12
29 71
8 9
47 53
13 4
77 23
3 14
18 82
Clinicopathological features of 17 CRC patients Clinicopathological features of 17 CRC patients [11 men (64%) and six women (36%)] were recorded. The average age was 54.7 years (range: 30e72 years). Table 1 shows the clinicopathological characteristics of these 17 patients, all of whom met the stage IeIII CRC criteria of the Union for International Cancer Control.
Expression of 15 candidate genes in CRC patients after treatment with CCRT We compared the variations of gene expressions in the tissues of the 17 patients prior to and after treatment with CCRT. If the expression of a candidate gene was positive prior to treatment and became negative after treatment with CCRT, we defined the gene as being downregulated after treatment. By contrast, if the expression of a candidate gene was negative prior to treatment and became positive after treatment with CCRT, we defined the gene as being upregulated after treatment. Of the 15 candidate genes, CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2 genes were differentially downregulated in, respectively, 52.9%, 52.9%, 52.9%, 29.4%, and 29.4% of the total patients after treatment with CCRT. One gene, MET, was upregulated in 23.5% of the total patients after treatment with CCRT (Table 2). Although OLFM4 and hTER genes showed a higher proportion in downregulated cases after treatment with CCRT, they did not show differential proportion in upregulated cases.
Discussion Preoperative radiotherapy has widely been used to improve local control of disease and survival of patients following
treatment of rectal cancer. There is great variability, however, in the CCRT responses of different individuals due to the lack of knowledge of how radiation-responsive genes are induced or repressed and how cells deal with radiation exposure.9 Therefore, we developed markers for predicting the effectiveness of CCRT in CRC patients. In the present study, we demonstrated the overexpression patterns in CRC patients prior to and after
Table 2 Gene expression of 15 target genes using gene chip in 17 colorectal cancer patients treated with CCRT. Gene
Downregulated cases after CCRT (N Z 17)
Upregulated cases after CCRT (N Z 17)
CK-20 ELAVL4 EV12B TM4SF3 ATPA2 OLFM4 hTERT CD55 S100B KCTD2 CEA KLK7 CK-19 MUC1 MET
9 9 9 5 5 9 7 4 3 3 2 2 1 1 0
0 2 2 0 1 4 4 4 0 1 2 2 2 2 4
(52.9) (52.9) (52.9) (29.4) (29.4) (52.9) (41.1) (23.5) (17.6) (17.6) (11.7) (11.7) (5.8) (5.8) (0)
Data are presented as n (%). CCRT Z concurrent chemoradiotherapy.
(0) (11.7) (11.7) (0) (5.8) (23.5) (23.5) (23.5) (0) (5.8) (11.7) (11.7) (11.7) (11.7) (23.5)
166 treatment with CCRT. Of the 15 CRC-related genes, five genes (CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2) had a downregulation rate of >29.4% of the total patients after treatment with CCRT. Additionally, one gene (MET ) had an upregulation rate of >20% of the total patients after treatment with CCRT. These differentially expressed genes show potential to be predictive markers for CCRT efficacy. Both upregulation of CK-20, ELAVL4, EV12B, TM4SF3, and ATPA2 genes that can be detected in the tumor of patients prior to CCRT and upregulation of MET gene that can be detected in the tumor after treatment with low-dose radiation can indicate the radiosensitivity. Through either kind of detection methods, it can provide the predictive results of treatment efficacy for the clinician prior to applying the CCRT to patients. However, because of the small sample size analyzed in this study, there was no statistically significant correlation between the expression of the 15 genes and the clinicopathological features of the CRC patients. In addition, upand downregulation rates of the 15 genes in the CRC patients after treatment with CCRT were not very high. Higher patient numbers would help increase the sensitivity in detecting any differences, if they exist. Therefore, further investigation should be conducted with a large number of samples to verify any potential clinical value. It is hoped that the study results can help clinicians plan an effective course of treatment.
H.-J. Wu et al.
3.
4.
5.
6.
7.
8.
9. 10.
Conflicts of interest The authors declare no conflicts of interest. 11.
References 1. Swedish Rectal Cancer Trial. Improved survival with preoperative radiotherapy in resectable rectal cancer. N Engl J Med. 1997;336:980e987. 2. Gerard A, Buyse M, Nordlinger B, et al. Preoperative radiotherapy as adjuvant treatment in rectal cancer: final results of
12.
a randomized study of the European Organization for Research and Treatment of Cancer (EORTC). Ann Surg. 1988;208: 606e614. Goldberg PA, Nicholls RJ, Porter NH, et al. Long-term results of a randomised trial of short-course low-dose adjuvant preoperative radiotherapy for rectal cancer: reduction in local treatment failure. Eur J Cancer. 1994;30:1602e1606. Frykholm GJ, Pahlman L, Glimelius B. Combined chemo- and radiotherapy vs. radiotherapy alone in the treatment of primary, nonresectable adenocarcinoma of the rectum. Int J Radiat Oncol Biol Phys. 2001;50:427e434. Bosset JF, Calais G, Mineur L, et al. Enhanced tumorocidal effect of chemotherapy with preoperative radiotherapy for rectal cancer: preliminary resultsdEORTC 22921. J Clin Oncol. 2005;23:5620e5627. Hendlisz A, Van den Eynde M, Peeters M, et al. Phase III trial comparing protracted intravenous fluorouracil infusion alone or with yttrium-90 resin microspheres radioembolization for liver-limited metastatic colorectal cancer. J Clin Oncol. 2010; 28:3687e3694. Huang MY, Wang JY, Chang HJ, et al. CDC25A, VAV1, TP73, BRCA1 and ZAP70 gene overexpression correlates with radiation response in colorectal cancer. Oncol Rep. 2011;25: 1297e1306. Amundson SA, Do KT, Vinikoor LC, et al. Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Cancer Res. 2008;68:415e424. Correa CR, Cheung VG. Genetic variation in radiation-induced expression phenotypes. Am J Hum Genet. 2004;75:885e890. Wang JY, Yeh CS, Chen YF, et al. Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer. Int J Mol Med. 2006;17: 737e747. Huang MY, Wang HM, Chang HJ, et al. Overexpression of S100B, TM4SF4, and OLFM4 genes is correlated with liver metastasis in Taiwanese colorectal cancer patients. DNA Cell Biol. 2012;3: 43e49. Huang MY, Wang HM, Tok TS, et al. EVI2B, ATP2A2, S100B, TM4SF3, and OLFM4 as potential prognostic markers for postoperative Taiwanese colorectal cancer patients. DNA Cell Biol. 2012;31:625e635.