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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1051-1056

Circulatingcell-free DNA as a biomarker in the serum of patients with colorectal cancer


1 Department of Medical Biochemistry, Menoufia University, Shebin Elkom, Egypt
2 Department of General Surgery, Menoufia University, Shebin Elkom, Egypt
3 Department of Clinical Oncology, Menoufia University, Shebin Elkom, Egypt

Date of Submission14-May-2017
Date of Acceptance11-Jun-2017
Date of Web Publication04-Apr-2018

Correspondence Address:
Amany M Wahb
Department of Medical Biochemistry, Menoufia University, Shebin Elkom
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_349_17

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  Abstract 


Objective
The aim was to study whether the concentration of circulating cell-free DNA (ccf-DNA) in serum could be used as a diagnostic biomarker for patients with colorectal cancer (CRC).
Background
Early diagnosis of CRC represents the best chance for cure. The present screening programs have limited sensitivity and specificity. Increased levels of ccf-DNA provide promising biomarker for several diseases including cancer. It has been postulated that tumor necrosis causes release of DNA of varying sizes which is in contrast to apoptosis of normal cells where smaller amount of DNA fragments are released.
Patients and methods
This study was carried out on 80 participants classified into the following groups: group I included 40 patients with CRC, group II included 20 patients with benign diseases in colon and rectum, and group III included 20 healthy controls. Laboratory investigations including carbohydrate antigen 19-9 serum level by enzyme-linked immunosorbent assay technique and quantitative analysis of ccf-DNA through detection of short (115 bp) DNA fragments in serum by real-time quantitative PCR by amplifying the Associative Location Unit (ALU) repeats (ALU-qPCR) were performed for all participants.
Results
The median absolute serum ALU115 levels in CRC group was significantly higher than those in intestinal polyp and normal control groups (P = 0.001)
Conclusion
It was concluded that serum DNA concentrations may be valuable in early diagnosis, monitoring of progression, and prognosis of CRC.

Keywords: carbohydrate antigen 19-9, circulating cell-free DNA, colorectal cancer


How to cite this article:
El Shafie MK, Safan MA, Abou Elnour ES, Habib MS, Rageh TM, Alhanafy AM, Wahb AM. Circulatingcell-free DNA as a biomarker in the serum of patients with colorectal cancer. Menoufia Med J 2017;30:1051-6

How to cite this URL:
El Shafie MK, Safan MA, Abou Elnour ES, Habib MS, Rageh TM, Alhanafy AM, Wahb AM. Circulatingcell-free DNA as a biomarker in the serum of patients with colorectal cancer. Menoufia Med J [serial online] 2017 [cited 2018 Oct 17];30:1051-6. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1051/229219




  Introduction Top


Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the universe and the third most common cause of cancer death among US men and women[1],[2].

In Egypt, like mostdeveloping countries, the incidence of CRC is lower than that of developed nations with western lifestyle[3].

The survival rate in patients with CRC is generally poor, on the account that most CRC cases are in the late stages at the time of diagnosis, losing the chance for access to timely and standard treatment. The 5-year survival rate of CRC as it might have been diagnosed in its early stages is 55% in developed nations and 40% in developing countries. Therefore, the key to CRC management may be early detection and diagnosis[4].

A sensitive assay that could accurately diagnose the onset of cancer using noninvasively collected clinical specimens is ideal for early detection. The earlier and more accurately the diagnostic biomarker predicts disease onset, the more valuable it becomes[5].

Various serum markers including carcinoembryonic antigen as well as carbohydrate antigen 19-9 (CA19-9) have been perceived as tumor markers for CRC. However, not all CRC cases can be diagnosed by carcinoembryonic antigen or CA19-9 alone owing to unstable detection and increased concentrations in benign diseases[6].

Circulating cell-free DNAs (ccf-DNAs) are extracellular nucleic acids discovered in cell-free plasma/serum of humans. Circulating extracellular DNA could be found in healthy persons, persons with nonmalignant diseases, as well as persons with different malignancies. It is probable that a significant proportion is bound to protein molecules, possibly nucleosomes[7].

Theoretically, circulating DNA is mostly released from degrading cells after cleavage by endonucleases that cut the chromatin under those essential nucleosomes, which conserves and protects them from proteolytic digestion in blood[8].

In healthy persons, ccf-DNA enters circulation through apoptosis of lymphocytes and other nucleated cells. Apoptosis has a different DNA 'ladder' pattern that shows particular banding at 200 bp that results from endonuclease-mediated double-strand cleavage between nucleosomes[9], whereas in patients with cancer, ccf-DNA most likely results from tumor necrosis, but other proposed mechanisms incorporate lysis of circulating tumor cells or of micrometastasis, or because of active release. Necrosis generates a range of DNA fragments with distinctive strand lengths, mostly large DNA fragments, owing to random and incomplete digestion of genomic DNA by DNAses[10].

ccf-DNA concentrations might be valuable in early integral diagnosis and monitoring of progression of CRC, and it was found that concentrations of ccf-DNA in patients with primary CRC might have been significantly higher than that in patients with intestinal polyps and healthy controls[11]. So, the aim of this study was to study whether the concentrations of ccf-DNA in serum can be used as a diagnostic biomarker for patients with CRC.


  Patients and Methods Top


This study was carried out in Medical Biochemistry, General Surgery and Clinical Oncology and Nuclear Medicine Departments, Faculty of Medicine, Menoufia University. It included 80 individuals: 60 patients and 20 age-matched and sex-matched healthy participants from May 2015 to December 2016. The studied participants were categorized into three groups.

Group I: it included 40 patients with histopathologically proven CRC (22 male and 18 female), and their ages ranged from 22 to 74 years, with mean age of 49.4 ± 13.5 years. Group II: it included 20 patients with benign diseases in colon and rectum. Group III: it included 20 age-matched and sex-matched healthy controls. An informed written consent was obtained from all participants, and the study was approved by the Ethical Committee of Medical Research, Faculty of Medicine, Menoufia University.

The exclusion criteria were patients with autoimmune diseases, tissue injury, viral diseases, or trauma at the time of examination.

All studied participants were subjected to full history taking, general clinical examination, abdominal ultrasound, computed tomography scan of the abdomen, colonoscopy, biopsy, histopathological examination (for patients only), metastatic workup including chest radiography and bone scan (for patients only), and laboratory investigations, which included detection of serum CA19-9 level by enzyme-linked immunosorbent assay (ELISA) and real-time ALU-qPCR to assess the concentration of serum ccf-DNA.

Initially, 5 ml of venous blood was withdrawn by venipuncture, transferred into plain tube, left to clot, and then centrifuged for 10 min at 4000 rpm. The serum obtained was stored at –80°C until analysis of serum CA19-9 and DNA extraction for quantitative detection of ccf-DNA.

Serum CA19-9 was determined using ELISA method, using Human CA19-9 ELISA kit (Chemux bioscience Inc., Minneapolis, USA, Canada) following the manufacturer's instructions[12].

Quantitative measurement of serum ccf-DNA was done by real-time PCR technique. It was done in two main steps. Genomic DNA was extracted from serum by GeneJET Viral DNA and RNA Purification Kit (Thermo Scientific, San Diego, USA)[13].

For the assessment of the concentration serum ccf-DNA short fragment (115 bp) from a consensus sequence with abundant genomic ALU repeats was amplified and quantified. The ALU-qPCR result obtained with ALU115 primers represents the total amount of serum DNA by using the 2x SensiFAST SYBR Lo-ROX kit (Bio Reagents Ltd., Chicago, USA) on Applied Biosystems 7500 Real-Time PCR systems.

The ALU115 primers were as follows: 5'-CCTGAGG TCAGGAGTTCGAG -3' (forward) and 5'-CCCGAGTAGCTGGGATTACA-3' (reverse). The reaction mixture was prepared by mixing 10 μl Master Mix, 1 μl of each primer (sigma), and 3 μl of DNAse-free water in a total reaction volume of 20 μl. For each unknown reaction, 5 μl (0.1 μg/μl) of DNA extract was added, and for the negative control reaction, 5 μl of DNAse-free water was added. The cycling parameters of ALU (115 bp) were as follows: initial denaturation step at 95°C for 3 min, one cycle of denaturation at 95°C, followed by 30 cycles of melting at 95°C for 5 s and annealing/collection at 62°C for 30 s using the Applied Biosystems 7500 software version 2.0.1.

The absolute amount of serum DNA in each sample was determined using a standard curve with serial dilutions (0.222–25 000 ng/ml) of human genomic DNA. A negative control was run with each reaction plate. Standard curve was made for ALU115 primer set through PCR amplifying 10-fold serially diluted human genomic DNA specimens[14].

Data were collected, tabulated, and statistically analyzed using an IBM personal computer with statistical package of the social sciences (SPSS) version 20.0 (2011; SPSS Inc., Armonk, New York USA) and Epi Info 2000 programs. Quantitative data were presented in the form of mean, SD, and range, and qualitative data were presented in the form of numbers and percentages. Analysis of variance test (f) was used for comparison of different groups with quantitative parameter variables. χ2-Test was used for qualitative variables. Mann–Whitney test (U) is used for quantitative nonparametric variables. Kruskal–Wallis test was used for groups of not normally distributed variables.

P value of less than or equal to 0.05 was considered statistically significant.


  Results Top


There was a statistically nonsignificant difference between the patients with CRC, those with benign disease, and control groups, as they were homogenous in terms of age and sex [Table 1].
Table 1: Demographic data of the studied groups

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There was a statistically nonsignificant difference between the studied groups regarding renal and hepatic functions.

There was a statistically significant difference in serum levels of CA19-9 and ALU115 between group I and both of groups II and III, whereas there was a statistically nonsignificant difference in serum levels of CA19-9 and ALU115 between groups II and III [Table 2].
Table 2: Statistical comparison among the studied groups regarding serum levels of carbohydrate antigen 19-9 and ALU115

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There was a statistically nonsignificant difference in ALU115 serum levels among patients with CRC regarding age, sex, site of the tumor, histopathological type of cancer, tumor grade, stage, size, or intestinal polyp [Table 3].
Table 3: Statistical comparison of ALU115 serum levels among the studied patients with colorectal cancer regarding demographic data, site, histopathologic type of the tumor, grading, staging, size of the tumor, and intestinal polyps

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The diagnostic accuracy for distinguishing patients with primary CRC from normal controls by ALU115 is 85%, with sensitivity 83%, specificity 90%, positive predictive value 96%, and negative predictive value 64% at a cutoff point of 426 ng/ml. The diagnostic accuracy for distinguishing patients with primary CRC from normal controls by CA19-9 is 59%, with sensitivity 60%, specificity 55%, positive predictive value 80%, and negative predictive value 31% at a cutoff point of 8.45 U/ml. Combined detection of ALU115 and CA19-9 improved the sensitivity to 88% [Table 4].
Table 4: Diagnostic validity of ALU115 and carbohydrate antigen 19-9 serum levels in diagnosis of cases of colorectal cancer

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There was a significant negative correlation between ALU115 serum levels and each of the size of tumor and the invasiveness of the primary tumor (T stage), whereas there was a significant positive correlation between ALU115 serum levels and the grade of tumor. There was a significant positive correlation between CA19-9 serum levels and each of the invasiveness of the primary tumor (T stage) and distant metastasis (M stage) [Table 5].
Table 5: Correlation coefficient between serum levels of ALU115 and carbohydrate antigen 19-9 and tumor characters among colorectal cancer group (n=40)

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  Discussion Top


CRC is a very heterogeneous disease. Many factors like molecular and genetic characteristics of the tumor determine the prognosis and response to treatment[15]. Screening in CRC is important for diagnosis of precancerous adenomatous polyps regarded as fundamental for prevention[16].

ccf-DNA quantification is a perfect candidate biomarker for early detection and prognosis monitoring for a blood-based CRC diagnosis test as it contains DNA released from dead cancer cells[17]. In this way, the present study was aimed at evaluating the concentrations of ccf-DNA in serum as a diagnostic biomarker for patients with CRC.

In this study, there was no significant statistical difference between the three studied groups regarding age. This is in agreement with Heitzer et al.[18] and Khan et al.[19].

Regarding sex, the current study showed a nonsignificant statistical difference between the studied groups. The percentages of males and females were 55 and 45%, respectively, in the patients with CRC and 55 and 45%, respectively, in healthy controls.

This is in agreement with Heitzer et al.[18] and Kim et al.[20] where the percentages of male and female were 57.9 and 42.1%, respectively, in the cases group and 51.8 and 48.2%, respectively, in the control group, with no differences in sex distribution between the two groups.

The present findings revealed that there was a statistically significant difference between patients with CRC and both of benign and controls regarding serum levels of CA19-9. However, there was no statistically significant difference in the level of CA19-9 between benign and controls.

This is matched with Wang et al.[21] and Mourtzikou et al.[22] who found a significant increase in CA19-9 levels in patients with cancer when compared with control. Moreover, Qi et al.[23] stated that CA19-9 levels proposed to differentiate benign from malignant colorectal diseases.

In contrast to our results, Al-Shuneigat et al.[24] reported no significant difference in the levels of CA19-9 in patients when compared with controls.

In the present study, there was a statistically significant difference in ALU115 serum levels in patients with cancer when compared with both controls and participants with benign disease, whereas there was a nonsignificant statistical difference in ALU115 serum level between benign and controls. This could be because of the exclusion of patients with diseases that might increase ccf-DNA concentrations such as autoimmune or viral diseases.

This result is similar to Da Silva et al.[25], who demonstrated a significant increase in ALU115 serum level in patients with cancer when compared with controls, and Hao et al.[8], who stated that a significant increase in ALU115 serum levels was found in patients with cancer when compared with benign group. In addition, Zaher et al.[26] and Bedin et al.[27] found that there was a nonsignificant statistical difference in the short fragment serum levels between benign and controls.

On the contrary, Hao et al.[8] reported that a statistically significant difference in ALU115 serum levels was found between benign and controls.

Discrepancies across different studies might be related to many factors, including the choice of participants or preanalytical and analytical techniques, such as type of sample, DNA isolation, or the quantification methods. These variables need to be standardized for interlaboratory analysis and reporting. Nevertheless, raised ccf-DNA levels can be detected in several other conditions in addition to those with malignancies[27].

Our results did not reveal any significant relations between age, sex, and clinicopathological parameters (e.g., site of tumor, histopathological type of cancer, tumor size, stage, and grade) and total ccf-DNA concentration levels in serum. This is in agreement with Gormally et al.[28].

Hao et al.[8] reported that ccf-DNA was found to be significantly related with age, histologic differentiation, and tumor stage (TNM) in patients with primary CRC.

Our results did not show any significant associations between age, sex, and clinicopathological parameters and the levels of CA19-9.

This is in accordance with Al-Shuneigat et al.[24] who reported that there was no significant relation between levels of serum CA19-9 and the differentiation of the tumor.

In contrast, Qi et al.[23] reported that there was a significant relation between pathologic stage and CA19-9, so it could be used for pathologic staging.

Our results revealed that there was a significant negative correlation between ALU115 serum levels and each of the size of tumor and the invasiveness of the primary tumor (T stage). There was a significant positive correlation between ALU115 serum levels and the grade of tumor.

This is matched with Bedin et al.[27] who reported that there was a significant positive correlation between total ccf-DNA concentration in the form of short fragment and histopathological grade.

In the current study, there was a significant positive correlation between CA19-9 serum levels and each of the invasiveness of the primary tumor (T stage) and distant metastasis (M stage).

Polat et al.[29] found that CA19-9 levels were significantly elevated in the presence of distant metastasis, so CA19-9 was found to be related to poor prognosis.

In the present study, the diagnostic accuracy for distinguishing patients with primary CRC from normal controls by ALU115 is 85%, with sensitivity 83%, specificity 90%, positive predictive value 96%, and negative predictive value 64%, at a cutoff point of 426 ng/ml. These results are in accordance with Hao et al.[8].

In the present study, the diagnostic accuracy for distinguishing patients with primary CRC from normal controls by CA19-9 is 59%, with sensitivity 60%, specificity 55%, positive predictive value 80%, and negative predictive value 31% at a cutoff point of 8.45 U/ml. These results are in accordance with Qi et al.[24].

Our results showed that combined detection of ALU115 and CA19-9 improved the sensitivity to 88%.


  Conclusion Top


This study concluded that ccf-DNA markers, in the form of total serum ccf-DNA as quantitative cancer-particular alterations, offer an interesting prospect for a blood test screen in CRC with the capability of discriminating normal individuals from patients with benign polyps and CRCs.

ALU115 in serum is significantly higher in patients with primary CRC than those in patients with intestinal polyps and healthy controls. However, large-scale prospective studies are required to establish the clinical utility for this biomarker.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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