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ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 2  |  Page : 569-574

Cell-free DNA as a biomarker of breast cancer


1 Department of Clinical Pathology, Menoufia University, Menoufia, Egypt
2 Department of General Surgery, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission29-Mar-2017
Date of Acceptance16-May-2017
Date of Web Publication27-Aug-2018

Correspondence Address:
Marwa M. M. Omar
Department of Clinical Pathology, Menoufia University, Shebein El kom, Menoufia Governorate, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_239_17

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  Abstract 


Background
Circulating plasma cell-free DNA (CFDNA) is comprised of nucleic acids in fringe blood of healthy persons and patients with several diseases that start from cell death. Apoptotic tumor cells may discharge DNA into the peripheral blood, and this may hold possibilities for the early detection of breast cancer.
Objective
This study aimed to assess CFDNA (glyceraldehyde 3-phosphate dehydrogenase) concentration in patients with malignant and benign breast lesions and healthy controls to investigate its role as a noninvasive marker for early detection of breast cancer.
Patients and methods
This study included 50 patients divided into three groups: group I included 30 newly diagnosed breast cancer female patients; group II included 10 female patients with benign breast lesions; and group III included 10 individuals of apparently healthy female individuals. CFDNA was extracted from plasma using NucleoSpin Plasma XS Kit. Concentration of CFDNA (glyceraldehyde 3-phosphate dehydrogenase) was measured using QuantiTect Probe PCR Kit by real-time PCR.
Results
The results revealed that there was a highly significant difference in the mean level of CFDNA cycle threshold between the malignant breast lesions and each of the benign lesions (34.31 vs. 40.30, P= 0.001) and controls (34.31 vs. 41.25, P= 0.001). However, there was no significant statistical difference between benign lesions and controls. Our study provides valuable data on the utilization of the concentration of free DNA for breast cancer recognition.
Conclusion
CFDNA level in plasma was observed to be higher in patients with breast cancer. It may have potential for the early identification of breast cancer.

Keywords: breast cancer, cell-free DNA, real-time polymerase chain reaction


How to cite this article:
El Edel RH, El Gamaal AS, El Said HH, Noreldin RI, Omar MM. Cell-free DNA as a biomarker of breast cancer. Menoufia Med J 2018;31:569-74

How to cite this URL:
El Edel RH, El Gamaal AS, El Said HH, Noreldin RI, Omar MM. Cell-free DNA as a biomarker of breast cancer. Menoufia Med J [serial online] 2018 [cited 2018 Nov 20];31:569-74. Available from: http://www.mmj.eg.net/text.asp?2018/31/2/569/239724




  Introduction Top


The breast is the most widely recognized site of tumor in ladies around the world [1]. Breast cancer is still the most incessant malignant tumor in ladies worldwide with nearly 1.7 million new cases diagnosed in 2012, representing 25% of all new female cancer cases [2]. In Egypt, the rate of breast cancer is higher than the overall records speaking of 32.04% of cancers in female individuals diagnosed during the period 2008–2011 [3]. Early identification or diagnostic tools such as breast mammography or ultrasound may have the potential to detect breast cancer [4]. However, these tools have limitations such as radiation exposure [5].

Serum carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) are two of the most broadly explored tumor markers. CEA and CA 15-3 are of limited use in early diagnosis because of the absence of specificity and affectability [6].

Circulating cell-free DNA (CFDNA) molecules were initially recognized in 1948. Ensuing investigations revealed CFDNA to be present in more elevated amounts among patients with autoimmune diseases and cancer as compared with that in healthy people [7].

CFDNA is comprised of extracellular nucleic acids found in cell-free plasma/serum of humans. There are a few terms being used, such as circulating nucleic acids, extracellular nucleic acids, or cell-free nucleic acids [8]. In healthy people, the concentration of circulating DNA is low, as most nonliving cells are expelled proficiently from circulation by phagocytes [9].

Apoptotic tumor cells may discharge DNA into the peripheral blood, and this may hold potential outcomes for the early recognition of cancer [10].

The utilization of DNA as a biomarker in clinical medicine for early diagnosis, prognosis, and observing of treatment has been a huge progression in the field [11].

The aim of this study was to quantitatively measure CFDNA concentration in patients with malignant breast cancer compared with that in patients with benign breast tumors and healthy controls. In addition, we also aimed to investigate its role as a noninvasive marker for breast cancer detection.


  Patients and Methods Top


This study was carried out at the Clinical Pathology Department, Faculty of Medicine, Menoufia University, and at Clinical Biochemistry Department, National Liver Institute, Menoufia University, in the period between May 2014 and January 2016. This study included 50 patients. The studied patients were divided into three groups. Group I included 30 newly diagnosed breast cancer female cases, and their ages ranged from 32 to 62 years. Group II included 10 female patients with benign breast lesions, and their ages ranged from 33 to 52 years. Group III included 10 female individuals of apparently healthy and age-matched controls, and their ages ranged from 39 to 60 years.

All patients enrolled in this study were newly diagnosed breast cancer patients with no surgery; no chemotherapy had to be started after diagnosis.

The study protocol was approved by the Ethical Committee in Menoufia University. Informed consents were taken from the patients before the beginning of the study.

All participants were subjected to the following.

Full history taking, complete clinical examination, breast mammography for benign and malignant breast lesions, chest radiography for cancer patients, and liver and bone scan for cancer patients to exclude metastasis, and laboratory investigation including complete blood count, liver function tests, kidney functions, serum tumor markers CEA and CA 15-3, and estimation of CFDNA in plasma using real-time PCR.

Samples collection

A volume of 10 ml of venous blood was collected from all patients. The first part was collected in a plain vacutainer tube and left to clot at 37°C. Sera were separated by centrifugation and used for immediate assay of liver, kidney functions, and tumor markers (CEA and CA 15-3). The second part was collected in dipotassium EDTA tube for complete blood count. The third part was transferred into another (EDTA) tube and thereafter centrifuged for 10 min at 4000 rpm. The plasma was transferred to new Eppendorf tubes and centrifuged again at a maximum speed (16 000g) for 10 min to remove cellular DNA completely from the plasma fractions. Thereafter the DNA was extracted for the estimation of CFDNA.

Assay methods

DNA extraction

DNA extraction from plasma was performed using NucleoSpin Plasma XS Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany).

DNA amplification and detection was carried out using QuantiTect Probe PCR Kit. (Qiagen, Milan, Italy)

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in CFDNA was determined by real-time PCR using the QuantiTect Probe PCR Kit and the ABI 7500 Real-Time PCR System (Applied Biosystems, Centre Drive Foster City, CA, USA).

Measurement of free DNA concentration

The forward primer 5'-GGAAGGTGAA GGTCGG AGTC-3', reverse primer 5'-GAAGATGGTGA TGGGATTTC-3', and probe 5'-FAMCAAGCTTCCCGTTCTCAGCC-TAMRA-3' were used to amplify the sequence of the GAPDH gene. The real-time PCR mixture comprised 25 μl reaction mix, 12.5 μl QuantiTect Probe PCR Master Mix, 1 μl PCR forward primer, 1 μl reverse primer, 1 μl probe, 6.5 μl serum DNA, and 4 μl ddH2O. Cycle conditions were as follows: denaturing at 95°C for 5 min followed by 45 cycles of 95°C for 30 s, 60°C for 60 s, and a final extension at 72°C for 10 min. The result was expressed by cycle threshold, which is inversely proportional to CFDNA concentration.

Statistical analysis

The data collected were tabulated and analyzed by statistical package for the social science software (SPSS) for Windows statistical package (version 20; SPSS Inc., Chicago, Illinois, USA) and MedCalc 13 for Windows (MedCalc Software BVBA, Ostend, Belgium) on IBM-compatible computer; a value less than 0.05 was considered as significant.


  Results Top


There was a statistically significant difference between the breast cancer group and control group as regards CEA (P = 0.001), whereas, there was no statistically significant difference between the breast cancer group and benign group (P = 0.118). However, there was a statistically significant difference between the breast cancer group, benign group, and control group as regards CA 15-3 (P = 0.001), but there was no statistically significant difference between the benign breast lesion group and control group (P = 0.12) [Table 1].
Table 1: Statistical comparison of tumor markers between studied groups

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[Table 2] represents the level of plasma CFDNA (cycle threshold) in the breast cancer, benign, and control groups. The mean ± SD for the breast cancer group was 34.40 ± 3.03, ranging from 29.21 to 38.40, and for the benign and control groups it was 40.62 ± 1.15, ranging from 39.32 to 42.88, and 41.73 ± 2.03, ranging from 39.26 to 44.90, respectively. There was a highly statistically significant difference in the mean level of plasma CFDNA between the breast cancer group and each of the benign and control groups (P = 0.001), but there was no statistically significant difference between the benign and control groups (P = 0.343).
Table 2: Statistical comparison of cell-free DNA between studied groups

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[Table 3] and [Figure 1] represent the receiver operating characteristic curve for plasma CFDNA, CA 15-3, and CEA to differentiate between the breast cancer group and the benign breast lesions group. The results of CEA showed that the sensitivity was 40%, the specificity was 80%, the positive predictive value was 85.7%, and the negative predictive value was 30.8%. The results of CA 15-3 showed that the sensitivity was 76.67%, the specificity was 50.0%, the positive predictive value was 82.1%, and the negative predictive value was 41.7%. The results of CFDNA showed that the sensitivity was 86.67%, the specificity was 60.0%, the positive predictive value was 82.1%, and the negative predictive value was 60.0%.
Table 3: Receiver operating characteristic curve values for plasma cell-free DNA, cancer antigen 15-3, and carcinoembryonic antigen to differentiate between the breast cancer group and the benign breast lesions group

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Figure 1: Receiver operating characteristic curve for plasma cell-free DNA (CFDNA), cancer antigen. (CA) 15.3, carcinoembryonic antigen. (CEA) to differentiate between the breast cancer group and the benign breast lesions group.

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There were significant statistical differences between CFDNA and tumor grade, tumor stage, lymph node status, and metastasis, and no significant statistical difference between CFDNA and menopause and tumor type in the breast cancer group [Table 4].
Table 4: Relation between cell-free DNA and different parameters in the breast cancer group

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  Discussion Top


With over 1.3 million annually new diagnosed cases, breast cancer is the most widely recognized analyzed tumor in ladies around the world [12]. The CFDNA has been used as a strong diagnostic and prognostic marker because CFDNA is typically modified clinched alongside malignancies [13]. It is accepted that the sources of CFDNA in the blood stream are necrotic and apoptotic cells [14].

The aim of the present study was to assess CFDNA (GAPDH) concentration in patients with malignant and benign breast lesions and healthy controls to investigate its role as a noninvasive marker for breast cancer early detection.

The current study showed that on the assessment of free DNA, the content is dependent upon the identification of the GAPDH gene. GAPDH is one of the most commonly used housekeeping genes in molecular biology. It is one of the single-copy gene families in the human genome and is portrayed by low gene amplification or deletion mutation probability in different types of cancer occurrence. Thus, GAPDH will be an incredible selection to quantify the free DNA content released from cancers and to reflect the tumor load [15].

In the present study, we compared the levels of CEA between the breast cancer group, benign disease group, and the healthy control group. It was found that there was no significant statistical difference between the breast cancer group and the benign disease group (P = 0.118); however, there was a significant statistical difference between the breast cancer group and the control group (P = 0.001). Moreover, on comparing the levels of CA 15-3 between the breast cancer group, the benign disease group, and the healthy control group, it was found that there was a significant statistical difference between the breast cancer group and the benign disease group (P = 0.019); there was also a significant statistical difference between the breast cancer group and the control group (P = 0.001).

These results were in agreement with He et al. [6] who stated that serum CEA and CA 15-3 levels were raised in 37.0 and 45.6% of breast cancer patients, respectively. These results were similar to results in other studies (CEA: 36.0–50.7%, CA 15-3: 36.4–55.6%) [16].

At present, there will be a discussion with regard to the utilization of CEA and CA 15-3 in the diagnosis of breast cancer. The European Society for Medical Oncology and the European Group on Tumor Markers suggested that schedule estimation of claiming tumor markers, for example, such as CEA and CA 15-3, ought be performed on patients with breast cancer [17]. However, the American Society of Clinical Oncology (ASCO) does not suggest schedule estimation of CEA, CA 15-3, or other tumor markers for patients with breast cancer [18]. In the present study, plasma CFDNA was significantly increased in the breast cancer group (computerized topography 29.21) when compared with that in both the benign group (CT 42.88) and the control group (CT 44.90) (P = 0.001 for both groups). These results are in concordance with the results of Hashad et al. [19]; they reported that levels of CFDNA in patients with breast cancer were significantly higher in comparison with the benign disease group and the healthy control group.

Moreover, other studies revealed elevated levels of CFDNA in breast cancer as reported by Cabral et al. [20] and Dawson et al. [21]; they stated that there was a significant elevation in CFDNA in malignant cases compared with that in nonmalignant cases. A possible reason may be that the CFDNA is discharged from cancer cells and is not found in the hyperplasia samples or in healthy controls.

In the present study there was no significant statistical difference between menopause and CFDNA (P = 0.05). In agreement with our findings, Lehner et al. [22] demonstrated that neither in premenopausal patients with breast cancer nor in young healthy ladies was there an impact on CFDNA levels.

In the present study there was significant statistical difference between the level of plasma CFDNA and tumor staging (P = 0.001). The level of CFDNA was significantly higher in stage III when compared with stages I and II, with P value of 0.008. In agreement with our results, Umetani et al. [23] watched a statistically significant increase in CFDNA in patients with breast cancer stages III and IV over patients with breast cancer stages I and II, with P value of 0.05, and they attributed this rise to more corruption happening in late tumor phases than early phases.

The level of CFDNA was significantly higher in breast cancer patients with lymph node metastasis over individuals without lymph node metastasis (P = 0.001). This was in concordance with Roth et al. [24]who showed that the level of CFDNA was significantly higher in lymph node-positive breast cancer patients than in patients without lymph node metastasis. This may be explained by the fact that the more the lymph node involvement, the more the corruption and apoptosis, and the more the DNA released into the circulation.


  Conclusion Top


In conclusion, this study revealed that the serum levels of CFDNA were significantly increased in patients with breast cancer, compared with those of patients with benign breast tumors and healthy controls. Furthermore, CFDNA levels were observed to increase as breast cancer progressed to later disease stages. Thus, quantitative identification of CFDNA may possess value for early identification of breast cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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



 

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