|Year : 2008 | Volume
| Issue : 2 | Page : 57-59
Utility of blood DNA levels in diagnosis of breast cancer
R Nalini1, CR Wilma Delphine Silvia2, Sheila Uthappa1
1 Department of Biochemistry and Biophysics, St. John’s Medical College, Bangalore, Karnataka, India
2 Department of Biochemistry, M.S.Ramaiah Medical College, Bangalore, Karnataka, India
|Date of Web Publication||1-Aug-2008|
C R Wilma Delphine Silvia
Department of Biochemistry, M.S. Ramaiah Medical College, MSR Nagar, Bangalore - 54, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Prognostic factors, including both histopathological and biochemical variables, influence the choice of modality and the course of therapy in breast cancer. The biomarkers found in biological fluids, particularly in blood, apparently hold the best promise for the development of screening assays.
Aim: To find out if any correlation exists between blood DNA level and tumor stage, size and grade.
Materials and Methods: This case-control study was carried out on 52 female patients in the age-group of 18-70 years. The cases comprised 25 patients with histopathologically confirmed malignant breast cancer, while 27 patients with benign breast tumors served as the control group.
Statistical Analysis: We used the Student's 't' test to compare the differences between the blood DNA levels in the two groups. Pearson's test was performed to find out the correlation between blood DNA levels and the TNM stage, tumor size and grade
Results: It was observed that blood DNA levels showed statistically significant correlation with the TNM stage, tumor size and grade.
Conclusion: The blood DNA level can be utilized as a noninvasive marker to assess tumor aggressiveness. Thus, it can be useful as a prognostic marker and as a marker of tumor burden.
Keywords: Breast cancer, DNA, tumor
|How to cite this article:|
Nalini R, Wilma Delphine Silvia C R, Uthappa S. Utility of blood DNA levels in diagnosis of breast cancer. J Can Res Ther 2008;4:57-9
Breast cancer accounts for approximately one-fourth of all cancers in women and is second only to cervical cancer as the leading cause of cancer deaths in women.  There are various modalities of treatment for breast cancer.  Of the surgical approaches, either breast-conserving or radical surgery is performed depending on the aggressiveness of the tumor. If it were possible to characterize the aggressiveness of the tumor before submitting the patient to surgery, it would help the surgeon to decide between the conservative and radical modes of treatment. Aggressiveness is assessed by means of many factors: the tumor size, its histopathologic type and grade, its multicentricity, DNA ploidy status, nodal status, and presence of metastasis. All these parameters are available only postoperatively. The concentration of nucleic acids in plasma or serum has important implications in the diagnosis, prognosis, and monitoring of many malignancies.  In breast cancer, it has been observed that even before the tumor becomes clinically apparent, tumor cells are present in the circulation. Many studies have been conducted that have sought to assay tumor-derived cells and DNA in the blood of breast cancer patients.  Tumor markers are used in cancer for diagnosis, staging, prognosis, and monitoring of therapy in malignancies. There are conflicting results reported on the correlation between DNA levels and tumor size and stage in breast carcinoma.  Hence, the present study aimed to find out the correlation between DNA levels in blood and the established tumor variables such as tumor size, grade, and stage.
| > Materials and Methods|| |
The study was carried out on 52 female patients with either benign or malignant breast tumors. There were 27 patients with malignant tumors (the cases); these patients had undergone surgery and the diagnosis had been histopathologically confirmed. The 25 women with benign tumors (the controls) had their diagnosis confirmed by detailed clinical evaluation and fine needle aspiration cytology. All patients were in the age-group of 18-70 years. None of the patients had diabetes, tuberculosis, generalized infections, liver or kidney disease. None of them were smokers or alcohol abusers. The study was conducted after informed consent was obtained from the subjects. Approval of the ethical committee of the institution was also obtained prior to the study.
Preoperatively, EDTA venous blood samples were collected with all aseptic precautions from both cases and controls. The DNA was extracted by salt-chloroform method.  The EDTA blood sample was mixed with lysis buffer (proteinase K, sodium chloride, EDTA, and sodium dodecyl sulphate) and incubated at 55°-65°C with periodic mixing. After incubation, saturated NaCl and chloroform were added; this was mixed thoroughly for 10 min by inverting the tube repeatedly and the mixture was then centrifuged. The upper aqueous phase was used for precipitation of DNA. The DNA was precipitated by ethanol  and estimated colorimetrically, using the diphenylamine method.  All the reagents used in the estimation were of analytical grade.
We used Student's ' t ' test to assess the significance of the difference between the mean blood DNA levels in benign and malignant cases. Pearson's correlation was used to study the relationship between the variables in the study.
| > Results|| |
Student's ' t ' test was performed to determine the significance of the difference between the mean levels of the variable in controls (benign) and cases (malignant). A cut-off value of 40 mg/l was used for statistical tests. 
From [Table 1] it can be seen that there is a significant difference between the mean blood DNA levels in the benign and malignant groups ( t = 7.17; P = 0.000). In addition, blood DNA levels were elevated to twice the cut-off value in 36% of malignant tumors.
In order to determine how the level of the marker changed with progressive clinical disease, we performed a correlation test of blood DNA level against TNM staging. There was a positive correlation of 0.81 between the level of the marker and the TNM stage, suggesting that the marker closely follows the clinical picture [Figure 1].
Pearson's test was also performed to ascertain if the blood DNA values correlated with tumor burden. We found a positive correlation between the marker and the tumor size (r = 0.69) [Figure 2]. A positive correlation was also seen between blood DNA values and tumor grade, with a correlation value of 0.51 [Figure 3].
| > Discussion|| |
Nucleic acid level in blood, particularly that of DNA, has assumed importance as a marker of malignancy. It has been found to be useful in the diagnosis, prognostication, and monitoring of cancer.
In our study, the mean blood DNA level was significantly higher in the group with malignant tumors as compared to the control group, with a t value of 7.17 and P = 0.000. This is similar to the findings obtained by Leon  and others, who reported a higher concentration of serum DNA in cancer patients when compared to normal individuals. With the cut-off at 40 mg/l, it is found that around 36% of patients with malignancy showed DNA levels more than twice the cut-off. The sensitivity and specificity of the marker in breast cancer has been found to be 92.0% and 88.9%, respectively, while the positive and negative predictive values were found to be 86.9% and 93.1%, respectively. This implies that, with a cut-off value of 40 mg/l, 86.9% of patients with elevated blood DNA values are likely to have malignancy. Similarly, 93.1% of patients with normal blood DNA levels are likely not to have malignancy. In summary, since blood DNA levels are found to be elevated in patients with malignant tumors, it has the potential to be a sensitive and specific marker in malignancy of the breast. Among patients with malignant tumors, the mean DNA level increased with the TNM stage, with a correlation coefficient of 0.81. As the TNM stage increases, the prognosis becomes poorer and, therefore, the blood DNA level could be used as a prognostic marker. As the size of the tumor increases, the blood DNA level also increases and thus serves as a marker of the tumor burden. There is also a positive correlation (r = 0.51) between the blood DNA level and the grade of differentiation of the tumor. Increase in tumor grade implies poor differentiation of the cells in the tumor.  Higher blood DNA levels are found in cases of higher grade, ie, tumors which are ill-differentiated. Such tumors carry a poor prognosis. Thus, DNA levels could also be used to predict the prognostic outcome in patients. The findings of this study are consistent with other studies on plasma DNA at the molecular level in cases of pancreatic carcinoma,  non-small-cell lung cancer,  melanoma,  and hepatocellular carcinoma. 
In the present study, DNA levels in plasma are significantly higher in patients with malignant breast tumor than in those with benign breast tumor with good analytical performance. DNA levels in blood correlate with histopathological variables like stage, size, and grade of the tumor in breast cancer, indicating that it has the potential to be a useful marker of tumor burden. The DNA concentration in blood obtained preoperatively could serve as a relatively noninvasive marker to assess tumor aggressiveness. Thus, the blood DNA level. which reflects the clinical status, could help the clinician to adopt the appropriate therapeutic measures.
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[Figure 1], [Figure 2], [Figure 3]