Article Text
Abstract
Aims The aim of this study was to report circulating cell-free DNA using ALU247 and ALU247/ALU115 biomarkers in serum of operated and non-operated patients with colorectal cancer (CRC).
Methods To undertake this, 90 blood samples were collected, including 30 samples from healthy volunteers; 27 samples from CRC non-operated patients and 33 samples from CRC-operated patients. Circulating cell-free DNA was verified through quantitative real-time PCR (qPCR) using ALU115 and ALU247 primers.
Results With regard to the ALU115-qPCR biomarker, the increased levels of circulating cell-free DNA in serum of non-operated patients were significant when compared with control (p<0.05). Moreover, levels of ALU247-qPCR biomarker were statistically significant between non-operated versus operated and non-operated versus control groups (p=0.000). With regard to the ALU247/115-qPCR biomarker, significant differences were observed between control versus non-operated patients (p=0.019), operated versus non-operated patients (p=0.005) and control versus operated patients (p=0.043).
Conclusions Thus, the ALU247 and ALU247/ALU115-qPCR biomarkers may be important in detecting and monitoring CRC patients in both early and late stages.
- Colorectal Cancer
- Molecular Pathology
- PCR
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Introduction
Colorectal cancer (CRC) represents the third most common cancer in men, and is the second leading cause of cancer in women worldwide.1 According to WHO, 1.2 million new cases of CRC were diagnosed, and more than 608 000 died in 2008.1
To date, it is well known that genetic and non-genetic factors are involved in the aetiology of CRC. Briefly, the tumour progression begins with the transformation of normal epithelium to neoplasia (adenoma), followed by invasive carcinoma and metastasis.2–4 The 5-year survival rate of CRC when it was diagnosed in its early stages, is 55% in developed countries and 40% in developing countries.5
Currently, screening tests to detect CRC are based on fecal occult blood test, stool DNA, sigmoidoscopy, colonoscopy and CT colonography.6 However, despite this wide range of screening tests, detection of dysplasia, or early stage CRC in asymptomatic patients, is limited due to the associated risks and low sensitivity, among other factors.6 For this reason, there is a need for non-invasive biomarkers that allow diagnostic treatment and care of patients in the early stage or late stage of CRC.
Recent studies have shown that raised levels of circulating cell-free DNA provide a promising biomarker for several diseases, including cancer.7–28 These studies are based on the circulating DNA in plasma or serum, in which the DNA of healthy patients is the target of an enzymatic treatment system during the process of apoptosis, leading to truncated DNA in 185 or 200 pb.20 On the other hand, during necrosis, apoptosis, autophagy and mitochondrial catastrophe, the tumour cells release DNA fragments of different sizes, which leads to raised levels of circulating cell-free DNA with longer fragments in serum or plasma.21 These studies have shown the existence of elevated levels of circulating cell-free DNA, probably released from cancer cells into the plasma or serum, which occurs in several types of cancer, such as lung,13 ,14 ,22 ,23 renal,9 prostate,28 ,29 breast,16 ,19 ,21 ,26 ,27 and CRC.15 ,18 ,20 ,25
Recent studies have been published of investigations using quantitative real-time PCR (qPCR) for ALU repeats (ALU-qPCR) in serum or plasma, as a promising biomarker of CRC.15 ,20 ,25 Although these studies agreed that the ALU247 and/or ALU247/ALU115 ratio may be a marker in CRC in the early stage or late stage, there have been no studies that report levels of ALU-qPCR in the serum of operated and non-operated CRC patients. In light of this, circulating cell-free DNA, using an ALU-qPCR marker could be a potential biomarker to detect and monitor both the early stage and late stage of CRC patients without the use of invasive methods.
Thus, the aim of this study was to report the levels of circulating cell-free DNA in serum using ALU115-qPCR, ALU247-qPCR and ALU247/115-qPCR biomarkers in operated and non-operated patients, when compared with the levels from healthy volunteers.
Materials and methods
Population
The blood samples were obtained from the Proctology Service of the Federal University of Alagoas State, Northeast Brazil; 90 volunteers agreed to participate in the study, and duly signed the consent forms; 30 patients without clinical significance or a family history of CRC, were included in the control group; 27 patients diagnosed with CRC without surgical treatment, were included in the non-operated group; and 33 patients who underwent surgical removal of the CRC, were included in the operated group. The stage of CRC was classified using the TNM staging system, according to the American Joint Committee on Cancer (AJCC). Patients with Stage I (3.70%), IIA (18.52%), II-B (7, 41%), IIIA (22.22%), IIIB (48.15%) were included in this study. All tumours were classified histologically as adenocarcinoma. The age of the participants ranged between 45 and 80 years. All patients were residents of the metropolitan area of Maceió, Alagoas State, Northeast Brazil. This study was approved by the Ethics Committee of the Federal University of Alagoas (UFAL), Northeast Brazil.
Processing of serum
A total of 4 mL of blood samples were collected from healthy, operated and non-operated patients. These samples were transferred to an 8 mL gel serum separator tube (BD—Becton, Dickinson and Company) and kept at room temperature for coagulation. Subsequently, the samples were centrifuged for 10 min at 1000 g. The samples were processed within 1–6 h after collection to prevent loss of cell-free DNA, as previously described by Umetani et al.20 The serum was filtered through acetate cellulose membrane and treated with 100 µL of tris-EDTA and 2 µL of proteinase K. The samples were placed in a water bath kept at 56°C for 1 h. After that, the samples were heated at 100°C for 8 min and centrifuged for 5 min at 10 000 g.
PCR-amplified microsatellite
PCR-based microsatellite markers were performed to confirm the absence of genomic DNA in serum samples. Thus, PCR was conducted to amplify the gene fragments in intron-1 of human tyrosine hydroxylase (TH01), lipoprotein lipase (LPL) and human fibrinogen α (FGA), using the primers listed in table 1. PCR was carried out in a total volume of 20 µL containing 50 ng of sample, 2 mM of MgCl2, 500 µM of each dNTP, 2 µM of specific primers and 1 unit of Taq DNA polymerase. The samples were processed in 30 cycles, with denaturation at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s and final extension at 72°C for 10 min. The PCR products were observed in 2.0% agarose gel with ethidium bromide.
Absolute quantification of ALU 115 and 247 primers
The serum samples were diluted 1:3 with ultrapure water, and 2 μL was used for the quantification of circulating cell-free DNA. The quantification was performed using real-time PCR with SYBRGreen Master Mix (Applied Biosystems) in accordance with the manufacturer's instructions. Absolute quantification of fragments was performed with amplification of ALU fragments using the following primers: ALU115 (F:5′-CCTGAGGTCAGGAGTTCGAG-3′; R5′-CCCGAGTAGCTGGGATTACA-3′) and ALU247 (F5′-CCTGAGGTCAGGAGTTCGAG-3′; R5′-CCCGAGTAGCTGGGAT TACA-3′), as previously described by Umetani et al.20 The ALU115 primers amplify both short and long fragments of DNA, while the ALU247 primers amplify the long DNA fragments, released from non-apoptotic cells. Real-time PCR was performed in both ALU115 and ALU247, using a total of 20 µL of template with 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, annealing at 64°C for 30 s and extension at 72°C for 30 s in PCR system 7500 standard (Applied Biosystems).20 The integrity of circulating cell-free DNA in serum was calculated by using ALU247/ALU115-qPCR ratio results.20
Statistical analysis
Data were expressed as mean±SD. Analysis of variance and Welch tests were conducted to compare the means of circulating cell-free DNA between healthy, non-operated and operated patients, while Dunnet's T3 test was used for multiple comparisons between the groups.
Results
No fragments were detected from the microsatellite markers, which makes it possible to confirm the absence of genomic DNA in all the serum samples (data not shown).
The ALU115-qPCR marker was used for the quantification of circulating cell-free DNA in healthy volunteers, and CRC non-operated and operated patients. The ALU115-qPCR biomarkers quantify the absolute levels of circulating cell-free DNA in serum originating from the apoptotic process. The mean levels of circulating cell-free DNA in serum from healthy patients using the ALU115-qPCR marker was 26.11 pg (SD 20.01). The mean levels of circulating cell-free DNA in the operated and non-operated patient groups were 62.10 pg (SD 59.43) and 186.71 pg (SD 106.25), respectively (table 2). The elevated levels of circulating cell-free DNA in the serum of non-operated patients was significantly high when compared with control (p<0.05). However, no statistical differences were observed in the levels of circulating cell-free DNA between operated versus non-operated, and operated versus control group (figure 1B).
The ALU247-qPCR marker allows quantification of circulating cell-free DNA in serum originating from tumour cells. The mean of the levels of circulating cell-free DNA in serum from healthy patients using ALU247-qPCR marker was 0.39 (SD=0.37) (log10 of pg/µL). In the case of the operated and non-operated patient groups, the means of the levels of circulating cell-free DNA in serum using ALU247-qPCR marker were 0.36 (SD=1.4) and 14.57 (SD=4.73), respectively (table 2). The elevated levels of circulating cell-free DNA in the serum of the non-operated group was statistically significant when compared with the control group (p=0.000). Moreover, the high level of circulating cell-free DNA in the serum of non-operated patients was statistically significant when compared with the operated group (p=0.000). However, no statistical differences were observed with regard to the means of circulating cell-free DNA in the serum of operated versus control patients (p=0.08) (table 2; figure 1A).
The ALU247/115-qPCR allows quantification of the integrity of circulating cell-free DNA in serum. The mean of the levels of cell-free DNA in serum from healthy patients using the ALU247/115-qPCR ratio, was 0.01 (SD=0.05) (log10 of pg/µL). The means of the levels of circulating cell-free DNA using the ALU247/115-qPCR ratio of operated and non-operated patients were 0.010 (SD 0.013) and 0.08 (SD 0.247), respectively. Moreover, the mean of circulating cell-free DNA of the ALU247/115-qPCR ratio was statistically significant between non-operated versus control (p=0.019), operated versus non-operated (p=0.005) and control versus operated patients (p=0.043) (table 2; figure 1C).
Discussion
This study reported the average levels of circulating cell-free DNA in healthy volunteers and CRC patients. To the best of our knowledge, this is the first study that has found increased levels of circulating cell-free DNA in serum of CRC operated and non-operated patients through quantification of ALU repeats. This study showed that elevated levels of ALU247-qPCR biomarker were significantly high in CRC patients when compared with those of normal volunteers. Moreover, this study showed that the ALU247/ALU115-qPCR biomarker was significantly higher in non-operated than operated CRC patients and the control group. Furthermore, the ALU247/ALU115-qPCR biomarker was significantly higher in operated patients than the volunteers without the disease. Thus, the ALU247-qPCR and ALU247/ALU115-qPCR biomarkers that were quantified using serum directly as a template could be particularly useful to detect CRC and to monitor CRC postoperative patients.
Several studies have been published using quantification of the circulating cell-free DNA as a promising biomarker in a number of cancers,7–28 including CRC.15 ,18 ,20 ,25 However, the differences in methodology and the lack of standardisation in these methodologies, have hampered the implementation of these tumour markers in clinical practice. For instance, several studies use plasma to quantify the circulating cell-free DNA, while other studies use serum as a template. Moreover, some studies performed DNA extraction30 and measured the levels of circulating cell-free DNA by qPCR, while other studies use serum20 or plasma15 as a direct template to quantify cell-free DNA. In this study, quantification of circulating cell-free DNA was conducted by using serum as a direct template.
In this study, it was shown that increased levels of ALU247-qPCR were found in CRC patients. The results are in accordance with those of other studies of circulating cell-free DNA, where plasma or serum were used.15 ,20 ,31
Other studies have shown that there are high levels of circulating cell-free DNA in CRC patients during recurrence and metastasis.30 ,32 This study demonstrated that ALU247/ALU115-qPCR could be a promising biomarker to use in simple blood tests and allow postoperative patients to be monitored.
In conclusion, the ALU247-qPCR and ALU247/115-qPCR could be useful biomarkers to use in blood tests as a means of detecting CRC in patients. Moreover, the ALU247/115-qPCR biomarker may offer a promising approach for monitoring CRC in operated patients. However, further investigations should be carried out in a large sample size to employ the method described in this study in preoperated and postoperated CRC patients.
Take-home messages
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ALU247/ALU115-qPCR biomarker was significantly higher in non-operated than operated colorectal cancer (CRC) patients and the control group.
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ALU247/ALU115-qPCR may be a potential biomarker to detect and monitor both the early stage and late stage of CRC patients.
References
Footnotes
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Contributors BFSF conducted all the experiments; APADG wrote the manuscript; MAdFLN and DAdA were involved in data collection and participated in some of the experiments; ACdF and LAFS participated in the design of the study and the review of the manuscript; JdCSN is the supervisor of this study and reviewed the manuscript. All the authors read and approved the final manuscript.
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Funding This study was supported by the Brazilian Federal Agency for the Support of Postgraduate Education (CAPES).
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Competing interests None.
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Patient consent Obtained.
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Ethics approval Approved by Ethics Committee of the Federal University of Alagoas (UFAL), Northeast Brazil.
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Provenance and peer review Not commissioned; externally peer reviewed.