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KLK11 mRNA expression predicts poor disease-free and overall survival in colorectal adenocarcinoma patients
Background: Dysregulated expression of several KLK family members has been observed in colorectal adenocarcinoma. In the present study, the prognostic value of KLK11 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma was examined. Materials & methods: Using quantitative real-time PCR, KLK11 mRNA expression was studied in 120 cancerous and 41 paired noncancerous colorectal specimens obtained from 120 patients with primary colorectal adenocarcinoma. Results: A significant upregulation of KLK11 transcripts in colorectal tumors was observed. KLK11 mRNA expression was associated with the depth of tumor invasion and the histological grade. Furthermore, KLK11 mRNA expression predicted poor disease-free and overall survival, independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy and chemotherapy treatment. Conclusion: KLK11 mRNA expression could be considered as a new molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients with highly invasive tumors and/or positive lymph nodes.
Dimitra K Alexopoulou1, Christos K Kontos1, Spyridon Christodoulou2, Iordanis N Papadopoulos2 & Andreas Scorilas*,1 Department of Biochemistry & Molecular Biology, University of Athens, Panepistimiopolis, 15701, Athens, Greece 2 Fourth Surgery Department, University of Athens, University General Hospital ‘Attikon’, 1 Rimini St, 12462, Haidari, Athens, Greece *Author for correspondence: Tel.: +30 210 727 4306 Fax: +30 210 727 4158 [email protected] 1
Keywords: biological tumor markers • colon cancer • kallikreins • prognostic biomarkers • quantitative real-time PCR
Colorectal cancer (CRC) constitutes the third most commonly diagnosed malignancy worldwide, and also the fourth most common cause of cancer death [1] . Current CRC screening tests can detect both primary CRC and relapse after surgery earlier than in the past decades, and are hence critical for the effective treatment and for the improvement of clinical outcome in patients with CRC. The American Society of Clinical Oncology Tumor Marker Panel and the European Group on Tumor Markers recommend quantification of preoperative and postoperative circulating CEA, as it constitutes an independent prognostic factor. Additionally, CEA is used in the clinical practice to monitor disease recurrence after surgical resection, as well as the response of CRC patients with metastatic disease to systemic therapy. However, neither CEA nor any of the other molecules that have been suggested as tumor biomarkers in the past, including cancer
10.2217/BMM.13.151 © 2014 Future Medicine Ltd
antigen 19-9 (CA19-9), possess remarkable sensitivity [2,3] . Although colonoscopy retains its place as the method of choice for CRC screening [4] , reliable prognostic and response-predictive tumor biomarkers are needed to decrease the future CRC burden of morbidity and mortality and to increase long-term survival. The gene family of KLKs consists of 15 members (KLK1–KLK15), tightly clustered in a tandem array spanning approximately 300 kb on chromosome 19q13.4 and represents the largest contiguous cluster of peptidase-encoding genes within the entire human genome [5–7] . KLK family members are serine peptidases implicated in cancer and have many different substrates, including PARs [8–10] . PARs compose a protein subfamily consisting of four G-protein–coupled receptors, two of which (PAR1 and PAR2) are aberrantly expressed in gastrointestinal malignancies [11–13] . Proteolytic cleavage
Biomarkers Med. (2014) 8(5), 671–685
part of
ISSN 1752-0363
671
Research Article Alexopoulou, Kontos, Christodoulou, Papadopoulos & Scorilas of PARs results in coupling of these receptors to heterotrimeric G proteins, and therefore to signal transduction, in many distinct cellular contexts [14,15] . For instance, enhanced expression and activation of PAR1 was shown to promote cell proliferation and motility in human colon cancer cells [16] . With regard to KLK11, there is no information regarding its proteolytic role in colorectal cancer. In breast cancer cells, this peptidase was shown to degrade IGFBP3, thus leading to an increase of IGF bioavailability, which in turn promotes cancer progression [17] . The prognostic potential of KLK family members in CRC has been intensively investigated so far [18,19] . Protein expression analysis of KLKs in cytosolic extracts from colorectal cancer tissue specimens and adjacent noncancerous colorectal mucosae demonstrated that KLK4, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK15 are aberrantly expressed in malignant colorectal tumors, compared with their noncancerous counterparts [20,21] . Furthermore, high KLK5, KLK6, KLK7, KLK13 and KLK14 protein levels predict poor outcome in patients with CRC. It is also noteworthy that the prognostic value of KLK5, KLK7 and KLK14 does not depend on patient age, TNM stage and tumor cell differentiation [20] . Moreover, protein levels of KLK11 in paraffin-embedded, rectal cancer tissue sections, as assessed by immunohistochemistry (IHC), are related to patients’ clinicopathological features, including histological differentiation, metastasis in regional lymph nodes, tumor invasion depth, clinical stage of the disease and CEA levels. Hence, immuno histochemical KLK11 positivity is an indicator of unfavorable prognosis for patients suffering from cancer in low rectum [22] . Furthermore, mRNA levels of several KLK genes are significantly elevated in CRC tissues than in their noncancerous counterparts, and usually display prognostic significance in this malignancy [19] . In this study, we analyzed the KLK11 mRNA expression in colorectal adenocarcinoma tissue samples and matched noncancerous colorectal specimens, using a newly developed, sensitive and cost-effective quantitative real-time PCR (qPCR) methodology, to further substantiate its prognostic value as a molecular tissue biomarker in colorectal adenocarcinoma. Materials & methods
672
Ethics Committee of the University General Hospital ‘Attikon’ (Athens, Greece). Moreover, it was carried out according to the ethical standards of the 1975 Declaration of Helsinki, as revised in 1996. Patients younger than 18 years of age, those who had already been treated by surgery, chemotherapy or radiation therapy for colorectal adenocarcinoma, and those who did not consent for the study were excluded. The routine preoperative work-up and postoperative management used in this surgical unit were not modified for the needs of the study. All surgical specimens of the colectomies were completely evaluated by a pathologist. Histopathological work-up has been guided by the need to establish accurate diagnosis, assess prognosis and plan further treatment of the patients. The macroscopic and microscopic examination of each specimen and the required IHC work-up, as was judged by the pathologist, were routinely reported. Clinicopathological data used for comparison in the present study such as the dimensions of the surgical specimens and the tumors, histological subtype, grade, tumor invasion of the resection margins, venous and lymphatic invasion, depth of tumor invasion (pT), lymph node count and metastasis (pN), distant metastasis (pM) and pTNM stage were also routinely reported. Decision for postoperative adjuvant chemoradiation was taken by oncologists. Finally, all the aforementioned data were recorded in an electronic database. Tissue specimens
Each surgical specimen was first opened-up and its luminal content was cleaned by rinsing normal saline. Under the observation of a pathologist, a sample from the tumor was obtained, avoiding macroscopically observed necrotic areas of the tumor. Each non cancerous colorectal tissue specimen was obtained from macroscopically observed normal intestine at the cut end of the surgical specimen and well away from the tumor. The cut ends of the surgical specimens were routinely processed by the pathologists to exclude invasion by the tumor. All samples were snap-frozen in liquid nitrogen next to resection of the tumors and stored at -85°C until further use.
Patients
Cancer cell line culture
This study included adult patients submitted to a colectomy for a primary colorectal adenocarcinoma in a single surgical unit (Fourth Surgery Department, University General Hospital ‘Attikon’, Athens, Greece) between 2000 and 2010. Informed consent was obtained from all participants in the current study, which was also approved by the institutional
The human breast ductal carcinoma cell line BT-474 was subcultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 kU/l penicillin, 0.1 g/l streptomycin, and 2 mM l-glutamine. Cells were seeded at a concentration of 4 × 105 cells/ml and incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2.
Biomarkers Med. (2014) 8(5)
future science group
KLK11 mRNA expression predicts poor disease-free & overall survival in colorectal adenocarcinoma patients
Total RNA isolation & cDNA synthesis
Colorectal adenocarcinoma tissue specimens were homogenized and then dissolved in TRI® Reagent (Life Technologies Ltd, CA, USA). Total RNA was isolated from pulverized specimens and subcultured BT-474 cells following the manufacturer’s instructions, dissolved in RNA Storage Solution (Life Technologies Ltd), and stored at -80°C. The concentration and purity of total RNA were determined by measuring the optical density at 260 and 280 nm. First-strand cDNA was synthesized from 2 μg of total RNA using the M-MLV Reverse Transcriptase (Life Technologies Ltd) and oligo-dT as primer. In more detail, the reaction mixture included 2 μg total RNA diluted in sterile, distilled water, 500 ng of oligo-dT primer, 4 μl of reaction buffer (5X, 250 mM Tris-HCl, pH 8.3 at 25°C, 375 mM KCl, 15 mM MgCl2, 0.1 M DTT), 0.5 mM each dNTP, 20 U of RNaseOUT™ (Life Technologies Ltd) RNase inhibitor, and 100 U of M-MLV Reverse Transcriptase. The final volume of the reverse transcription reaction was 20 μl. The initial reaction mix including total RNA, oligo-dT primer and dNTPs was incubated at 65°C for 5 min and then quickly chilled on ice. The final reaction mix was heated at 37°C for 50 min, and the reaction was stopped by thermal inactivation of the reverse transcriptase at 70°C for 15 min. Quantitative real-time PCR
qPCR was carried out using the SYBR Green chemistry in an Applied Biosystems 7500 Real Time PCR System (Life Technologies Ltd). The sequences of the KLK11 primers were 5´-CCTCTCCTCACGCTGTGTCA-3´ and 5´-GTTCTCACACTTCTGGTGCTCAA-3´, amplifying a common part (139 bp) of all known KLK11 splice variants. The sequences of the B2M primers were 5´-ACTGAATTCACCCCCACTGA-3´ and 5´-AAGCAAGCAAGCAGAATTTGG-3´, creating a 167-bp PCR product. The PCR mix contained 20 ng of cDNA, 5 μl Power SYBR Green PCR Master Mix (Life Technologies Ltd), and 2 μl of gene-specific primers (final concentration: 50 nM each). The final reaction volume was 10 μL. The cycling conditions are described here below: a denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and a final stage for the creation of a melting curve. Calculations of the comparative CT (2-ΔΔCT) method for KLK11 mRNA quantification & its experimental validation
Calculations were performed with the use of the comparative CT (2-ΔΔCT) method. The prerequisites for the use of the 2-ΔΔCT method [23] were experimentally validated by measuring CT values of KLK11 and B2M in a dilution series of BT-474 cDNA over a 104-fold range
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Research Article
and by plotting them against log cDNA dilution. The slopes of KLK11 and B2M amplification plots were -3.495 and -3.334, respectively. qPCR efficiency (E) for each amplicon was calculated based on the following formula: E = -1+10 (-1/α), where α is the slope of the respective standard curve. Consequently, the efficiencies of KLK11 and B2M amplification were 93.2 and 99.5%, respectively. B2M was used as an internal reference gene, as it has previously been shown that this is one of the best normalizing genes for comparing human colon primary carcinomas with their normal counterparts of the resection margin [24,25] . Moreover, the breast ductal carcinoma cell line BT-474 was used as a calibrator [23] . Normalized results were expressed as relative quantification units (RQU), standing for the ratio of KLK11 mRNA copies to B2M mRNA copies, measured in each colorectal tissue sample, in relation to the same ratio, determined for the calibrator. Study design
The clinicopathological data that were used in this retrospective study included tumor size, location, histological subtype, grade, venous and lymphatic invasion, depth of tumor invasion (T), presence of metastases in regional lymph nodes (N), distant metastases (M) and TNM stage. Determination of histological subtype and grade were based on WHO classification system (4th edition) [26] . Staging was done according to the TNM classification (7th edition) – which is the same for both clinical and pathologic staging – as designated by the American Joint Committee on Cancer (AJCC) to define colorectal cancer [27] . Postoperative radiotherapy and chemotherapy treatment information was available for 107 patients. Follow-up information was collected for 100 patients and included disease-free survival (DFS) and overall survival (OS) status, as well as the date of each event and the cause of death. Recurrence and death were considered as endpoints for DFS and OS, respectively. The maximum follow-up time was 10 years. Statistical analysis
For the comparison of the distributions of the KLK11 mRNA expression in the two groups of paired samples, we used the nonparametric Wilcoxon signed-rank test. It should be noted that KLK11 mRNA expression values below the detection limit of the quantification method were censored and substituted with a constant value, equal to half the detection limit. We also used the X-tile algorithm that generates optimal cutoff values while correcting for the use of minimum p-value statistics [28] , was used to create an optimal cutoff point for KLK11 mRNA levels in colorectal adenocarcinoma. Thus, the optimal cutoff value was
www.futuremedicine.com
673
Research Article Alexopoulou, Kontos, Christodoulou, Papadopoulos & Scorilas determined at 0.024 RQU, which is represented by the 36th percentile of the distribution of KLK11 mRNA expression. Based on this cutoff point, KLK11 mRNA levels were categorized as negative or positive. Then, potential associations between KLK11 mRNA expression status and clinicopathological variables were examined with the two-tailed chi-square (χ2) test, using also the Fisher correction, where appropriate. Correlations between KLK11 mRNA expression and the other continuous variables (age and tumor size) were assessed by Spearman correlation coefficient (rs). In order to assess the putative prognostic value of KLK11 mRNA expression, we built Kaplan–Meier DFS and OS curves, and used the log-rank (Mantel-Cox) test to evaluate any observed differences between the survival curves. So as to assess the association between the prognostic markers and the relative risks for recurrence and death from the specific disease, we created Cox proportional hazard regression models, after having tested the proportionality assumption. Moreover, multivariate Cox regression analysis included only those patients for whom the status of all variables was known. Multivariate Cox regression models were adjusted for patient gender, age, tumor size, location, histological subtype, grade, venous and lymphatic invasion, radiotherapy, and chemotherapy in both cases, as well as for depth of tumor invasion and nodal status in the case of DFS or for TNM stage in the case of OS. The level of significance in the current study was defined at a probability value
KLK11 mRNA expression predicts poor disease-free and overall survival in colorectal adenocarcinoma patients
Background: Dysregulated expression of several KLK family members has been observed in colorectal adenocarcinoma. In the present study, the prognostic value of KLK11 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma was examined. Materials & methods: Using quantitative real-time PCR, KLK11 mRNA expression was studied in 120 cancerous and 41 paired noncancerous colorectal specimens obtained from 120 patients with primary colorectal adenocarcinoma. Results: A significant upregulation of KLK11 transcripts in colorectal tumors was observed. KLK11 mRNA expression was associated with the depth of tumor invasion and the histological grade. Furthermore, KLK11 mRNA expression predicted poor disease-free and overall survival, independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy and chemotherapy treatment. Conclusion: KLK11 mRNA expression could be considered as a new molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients with highly invasive tumors and/or positive lymph nodes.
Dimitra K Alexopoulou1, Christos K Kontos1, Spyridon Christodoulou2, Iordanis N Papadopoulos2 & Andreas Scorilas*,1 Department of Biochemistry & Molecular Biology, University of Athens, Panepistimiopolis, 15701, Athens, Greece 2 Fourth Surgery Department, University of Athens, University General Hospital ‘Attikon’, 1 Rimini St, 12462, Haidari, Athens, Greece *Author for correspondence: Tel.: +30 210 727 4306 Fax: +30 210 727 4158 [email protected] 1
Keywords: biological tumor markers • colon cancer • kallikreins • prognostic biomarkers • quantitative real-time PCR
Colorectal cancer (CRC) constitutes the third most commonly diagnosed malignancy worldwide, and also the fourth most common cause of cancer death [1] . Current CRC screening tests can detect both primary CRC and relapse after surgery earlier than in the past decades, and are hence critical for the effective treatment and for the improvement of clinical outcome in patients with CRC. The American Society of Clinical Oncology Tumor Marker Panel and the European Group on Tumor Markers recommend quantification of preoperative and postoperative circulating CEA, as it constitutes an independent prognostic factor. Additionally, CEA is used in the clinical practice to monitor disease recurrence after surgical resection, as well as the response of CRC patients with metastatic disease to systemic therapy. However, neither CEA nor any of the other molecules that have been suggested as tumor biomarkers in the past, including cancer
10.2217/BMM.13.151 © 2014 Future Medicine Ltd
antigen 19-9 (CA19-9), possess remarkable sensitivity [2,3] . Although colonoscopy retains its place as the method of choice for CRC screening [4] , reliable prognostic and response-predictive tumor biomarkers are needed to decrease the future CRC burden of morbidity and mortality and to increase long-term survival. The gene family of KLKs consists of 15 members (KLK1–KLK15), tightly clustered in a tandem array spanning approximately 300 kb on chromosome 19q13.4 and represents the largest contiguous cluster of peptidase-encoding genes within the entire human genome [5–7] . KLK family members are serine peptidases implicated in cancer and have many different substrates, including PARs [8–10] . PARs compose a protein subfamily consisting of four G-protein–coupled receptors, two of which (PAR1 and PAR2) are aberrantly expressed in gastrointestinal malignancies [11–13] . Proteolytic cleavage
Biomarkers Med. (2014) 8(5), 671–685
part of
ISSN 1752-0363
671
Research Article Alexopoulou, Kontos, Christodoulou, Papadopoulos & Scorilas of PARs results in coupling of these receptors to heterotrimeric G proteins, and therefore to signal transduction, in many distinct cellular contexts [14,15] . For instance, enhanced expression and activation of PAR1 was shown to promote cell proliferation and motility in human colon cancer cells [16] . With regard to KLK11, there is no information regarding its proteolytic role in colorectal cancer. In breast cancer cells, this peptidase was shown to degrade IGFBP3, thus leading to an increase of IGF bioavailability, which in turn promotes cancer progression [17] . The prognostic potential of KLK family members in CRC has been intensively investigated so far [18,19] . Protein expression analysis of KLKs in cytosolic extracts from colorectal cancer tissue specimens and adjacent noncancerous colorectal mucosae demonstrated that KLK4, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK15 are aberrantly expressed in malignant colorectal tumors, compared with their noncancerous counterparts [20,21] . Furthermore, high KLK5, KLK6, KLK7, KLK13 and KLK14 protein levels predict poor outcome in patients with CRC. It is also noteworthy that the prognostic value of KLK5, KLK7 and KLK14 does not depend on patient age, TNM stage and tumor cell differentiation [20] . Moreover, protein levels of KLK11 in paraffin-embedded, rectal cancer tissue sections, as assessed by immunohistochemistry (IHC), are related to patients’ clinicopathological features, including histological differentiation, metastasis in regional lymph nodes, tumor invasion depth, clinical stage of the disease and CEA levels. Hence, immuno histochemical KLK11 positivity is an indicator of unfavorable prognosis for patients suffering from cancer in low rectum [22] . Furthermore, mRNA levels of several KLK genes are significantly elevated in CRC tissues than in their noncancerous counterparts, and usually display prognostic significance in this malignancy [19] . In this study, we analyzed the KLK11 mRNA expression in colorectal adenocarcinoma tissue samples and matched noncancerous colorectal specimens, using a newly developed, sensitive and cost-effective quantitative real-time PCR (qPCR) methodology, to further substantiate its prognostic value as a molecular tissue biomarker in colorectal adenocarcinoma. Materials & methods
672
Ethics Committee of the University General Hospital ‘Attikon’ (Athens, Greece). Moreover, it was carried out according to the ethical standards of the 1975 Declaration of Helsinki, as revised in 1996. Patients younger than 18 years of age, those who had already been treated by surgery, chemotherapy or radiation therapy for colorectal adenocarcinoma, and those who did not consent for the study were excluded. The routine preoperative work-up and postoperative management used in this surgical unit were not modified for the needs of the study. All surgical specimens of the colectomies were completely evaluated by a pathologist. Histopathological work-up has been guided by the need to establish accurate diagnosis, assess prognosis and plan further treatment of the patients. The macroscopic and microscopic examination of each specimen and the required IHC work-up, as was judged by the pathologist, were routinely reported. Clinicopathological data used for comparison in the present study such as the dimensions of the surgical specimens and the tumors, histological subtype, grade, tumor invasion of the resection margins, venous and lymphatic invasion, depth of tumor invasion (pT), lymph node count and metastasis (pN), distant metastasis (pM) and pTNM stage were also routinely reported. Decision for postoperative adjuvant chemoradiation was taken by oncologists. Finally, all the aforementioned data were recorded in an electronic database. Tissue specimens
Each surgical specimen was first opened-up and its luminal content was cleaned by rinsing normal saline. Under the observation of a pathologist, a sample from the tumor was obtained, avoiding macroscopically observed necrotic areas of the tumor. Each non cancerous colorectal tissue specimen was obtained from macroscopically observed normal intestine at the cut end of the surgical specimen and well away from the tumor. The cut ends of the surgical specimens were routinely processed by the pathologists to exclude invasion by the tumor. All samples were snap-frozen in liquid nitrogen next to resection of the tumors and stored at -85°C until further use.
Patients
Cancer cell line culture
This study included adult patients submitted to a colectomy for a primary colorectal adenocarcinoma in a single surgical unit (Fourth Surgery Department, University General Hospital ‘Attikon’, Athens, Greece) between 2000 and 2010. Informed consent was obtained from all participants in the current study, which was also approved by the institutional
The human breast ductal carcinoma cell line BT-474 was subcultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 kU/l penicillin, 0.1 g/l streptomycin, and 2 mM l-glutamine. Cells were seeded at a concentration of 4 × 105 cells/ml and incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2.
Biomarkers Med. (2014) 8(5)
future science group
KLK11 mRNA expression predicts poor disease-free & overall survival in colorectal adenocarcinoma patients
Total RNA isolation & cDNA synthesis
Colorectal adenocarcinoma tissue specimens were homogenized and then dissolved in TRI® Reagent (Life Technologies Ltd, CA, USA). Total RNA was isolated from pulverized specimens and subcultured BT-474 cells following the manufacturer’s instructions, dissolved in RNA Storage Solution (Life Technologies Ltd), and stored at -80°C. The concentration and purity of total RNA were determined by measuring the optical density at 260 and 280 nm. First-strand cDNA was synthesized from 2 μg of total RNA using the M-MLV Reverse Transcriptase (Life Technologies Ltd) and oligo-dT as primer. In more detail, the reaction mixture included 2 μg total RNA diluted in sterile, distilled water, 500 ng of oligo-dT primer, 4 μl of reaction buffer (5X, 250 mM Tris-HCl, pH 8.3 at 25°C, 375 mM KCl, 15 mM MgCl2, 0.1 M DTT), 0.5 mM each dNTP, 20 U of RNaseOUT™ (Life Technologies Ltd) RNase inhibitor, and 100 U of M-MLV Reverse Transcriptase. The final volume of the reverse transcription reaction was 20 μl. The initial reaction mix including total RNA, oligo-dT primer and dNTPs was incubated at 65°C for 5 min and then quickly chilled on ice. The final reaction mix was heated at 37°C for 50 min, and the reaction was stopped by thermal inactivation of the reverse transcriptase at 70°C for 15 min. Quantitative real-time PCR
qPCR was carried out using the SYBR Green chemistry in an Applied Biosystems 7500 Real Time PCR System (Life Technologies Ltd). The sequences of the KLK11 primers were 5´-CCTCTCCTCACGCTGTGTCA-3´ and 5´-GTTCTCACACTTCTGGTGCTCAA-3´, amplifying a common part (139 bp) of all known KLK11 splice variants. The sequences of the B2M primers were 5´-ACTGAATTCACCCCCACTGA-3´ and 5´-AAGCAAGCAAGCAGAATTTGG-3´, creating a 167-bp PCR product. The PCR mix contained 20 ng of cDNA, 5 μl Power SYBR Green PCR Master Mix (Life Technologies Ltd), and 2 μl of gene-specific primers (final concentration: 50 nM each). The final reaction volume was 10 μL. The cycling conditions are described here below: a denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and a final stage for the creation of a melting curve. Calculations of the comparative CT (2-ΔΔCT) method for KLK11 mRNA quantification & its experimental validation
Calculations were performed with the use of the comparative CT (2-ΔΔCT) method. The prerequisites for the use of the 2-ΔΔCT method [23] were experimentally validated by measuring CT values of KLK11 and B2M in a dilution series of BT-474 cDNA over a 104-fold range
future science group
Research Article
and by plotting them against log cDNA dilution. The slopes of KLK11 and B2M amplification plots were -3.495 and -3.334, respectively. qPCR efficiency (E) for each amplicon was calculated based on the following formula: E = -1+10 (-1/α), where α is the slope of the respective standard curve. Consequently, the efficiencies of KLK11 and B2M amplification were 93.2 and 99.5%, respectively. B2M was used as an internal reference gene, as it has previously been shown that this is one of the best normalizing genes for comparing human colon primary carcinomas with their normal counterparts of the resection margin [24,25] . Moreover, the breast ductal carcinoma cell line BT-474 was used as a calibrator [23] . Normalized results were expressed as relative quantification units (RQU), standing for the ratio of KLK11 mRNA copies to B2M mRNA copies, measured in each colorectal tissue sample, in relation to the same ratio, determined for the calibrator. Study design
The clinicopathological data that were used in this retrospective study included tumor size, location, histological subtype, grade, venous and lymphatic invasion, depth of tumor invasion (T), presence of metastases in regional lymph nodes (N), distant metastases (M) and TNM stage. Determination of histological subtype and grade were based on WHO classification system (4th edition) [26] . Staging was done according to the TNM classification (7th edition) – which is the same for both clinical and pathologic staging – as designated by the American Joint Committee on Cancer (AJCC) to define colorectal cancer [27] . Postoperative radiotherapy and chemotherapy treatment information was available for 107 patients. Follow-up information was collected for 100 patients and included disease-free survival (DFS) and overall survival (OS) status, as well as the date of each event and the cause of death. Recurrence and death were considered as endpoints for DFS and OS, respectively. The maximum follow-up time was 10 years. Statistical analysis
For the comparison of the distributions of the KLK11 mRNA expression in the two groups of paired samples, we used the nonparametric Wilcoxon signed-rank test. It should be noted that KLK11 mRNA expression values below the detection limit of the quantification method were censored and substituted with a constant value, equal to half the detection limit. We also used the X-tile algorithm that generates optimal cutoff values while correcting for the use of minimum p-value statistics [28] , was used to create an optimal cutoff point for KLK11 mRNA levels in colorectal adenocarcinoma. Thus, the optimal cutoff value was
www.futuremedicine.com
673
Research Article Alexopoulou, Kontos, Christodoulou, Papadopoulos & Scorilas determined at 0.024 RQU, which is represented by the 36th percentile of the distribution of KLK11 mRNA expression. Based on this cutoff point, KLK11 mRNA levels were categorized as negative or positive. Then, potential associations between KLK11 mRNA expression status and clinicopathological variables were examined with the two-tailed chi-square (χ2) test, using also the Fisher correction, where appropriate. Correlations between KLK11 mRNA expression and the other continuous variables (age and tumor size) were assessed by Spearman correlation coefficient (rs). In order to assess the putative prognostic value of KLK11 mRNA expression, we built Kaplan–Meier DFS and OS curves, and used the log-rank (Mantel-Cox) test to evaluate any observed differences between the survival curves. So as to assess the association between the prognostic markers and the relative risks for recurrence and death from the specific disease, we created Cox proportional hazard regression models, after having tested the proportionality assumption. Moreover, multivariate Cox regression analysis included only those patients for whom the status of all variables was known. Multivariate Cox regression models were adjusted for patient gender, age, tumor size, location, histological subtype, grade, venous and lymphatic invasion, radiotherapy, and chemotherapy in both cases, as well as for depth of tumor invasion and nodal status in the case of DFS or for TNM stage in the case of OS. The level of significance in the current study was defined at a probability value
