Circulating Cell-Free DNA Levels in Breast Cancer Patients
Circulating Cell-Free DNA Levels in Breast Cancer Patients
Objectives To evaluate circulating cell-free DNA (CFD) measured by a simple fluorescent assay as a biomarker of breast cancer.
Methods We enrolled 38 patients with breast cancer before surgery, two patients with noncancerous breast lesions, nine patients after surgery, 16 healthy participants, and 29 control women admitted to the hospital emergency ward and released without hospitalization. CFD levels were measured by a direct fluorescence assay.
Results Presurgery patients with cancer had elevated CFD levels (1,010 ± 642 ng/mL), which were higher than those measured in the healthy control group (395 ± 248 ng/mL, P < .001), the noncancer breast lesion group (386 ± 40 ng/mL), the nonhospitalized control group (492 ± 193 ng/mL, P < .001), and the postsurgery cancer group (398 ± 162 ng/mL, P < .01). The area under the receiver operating characteristic curve of the presurgery vs healthy patient group was 0.83. CFD levels correlated with tumor size (P = .03, ρ = 0.36), nodal involvement (P = .0003, ρ = 0.56), and TNM stage (P = .0002, ρ = 0.56). All patients with axillary node involvement had a CFD value greater than 600 ng/mL.
Conclusions CFD measured using a simple fluorometric assay has shown good correlation to stage and enhanced sensitivity to locally advanced disease. A large prospective study is warranted to evaluate if inclusion of this method as a decisive marker before mammography is advantageous.
Breast cancer is now the most common cancer diagnosed in women, and it is the leading cause of deaths from cancer among women in the United States. The incidence is approximately 1.3 million new cases a year, and an estimated 458,000 deaths were reported in 2008. Since 1990, mortality from breast cancer in industrialized countries has been decreasing due to advances in adjuvant therapy and the increasing use of screening mammography (reviewed in Warner).
The current screening of breast cancer is based on clinical breast examination and mammography. Since breast cancer is known to have an asymptomatic phase, mammography is an important tool in the early diagnosis of breast cancer.
Mammography is considered highly sensitive and specific for women older than 50 years. However, 20% of patients diagnosed with breast cancer are younger than 50 years, and for this group of younger women, a sensitive screening alternative is presently not available. Mammography screening is recommended every 2 years, and between examinations, lack of specificity and sensitivity precludes the routine use of all available serum markers of breast cancer screening. Notwithstanding the wide use of mammography for the detection of breast cancer, it is worth noticing that serious questions have been raised about the value of screening mammography. A recent large randomized prospective study from Canada estimated that 22% of mammography screens overdiagnosed invasive breast cancers. These findings indicate the urgent need for new markers that will not only allow the screening of young women and their follow-up between examinations but also afford the critical discrimination between benign and malignant disease.
The presence of elevated levels of circulating cell-free DNA (CFD) in the blood of patients with cancer was initially demonstrated by Leon et al in 1977. The easy accessibility of plasma and serum DNA has made these noninvasive approaches an appealing method to detect and follow up patients with cancer. Many studies have confirmed the initial observation of Leon and colleagues and found elevated CFD levels in patients with malignancies (reviewed in Gormally et al), including patients with breast cancer. The elevation in circulating CFD levels originates from the release of DNA from cellular necrosis and apoptosis of tumor cells and probably from dying cells in the surrounding tissue affected by the tumor.
Although there is general agreement on the value of CFD measurement for patients with cancer, there is no standard method for its routine clinical use. The currently available research methods for CFD measurement are work-intensive and expensive, and they require DNA extraction and realtime polymerase chain reaction (PCR). Consequently, CFD measurements are not used for routine patient management.
We have developed a convenient and simple DNA assay applied directly to biological samples. This assay is based on the use of the nucleic acid fluorochrome SYBR Gold (Invitrogen, Paisley, England) stain, which does not require prior processing of samples (ie, DNA extraction and amplification). The assay is performed by adding diluted fluorochrome to the samples and measurement of fluorescence. Using this assay, we measured CFD levels and followed tumor growth and rejection in mice and in patients with colorectal cancer and found that CFD levels are prognostic for disease progression and death.
The aim of the current study was to evaluate the potential of this new method for the detection and prognosis of patients with breast cancer.
Abstract and Introduction
Abstract
Objectives To evaluate circulating cell-free DNA (CFD) measured by a simple fluorescent assay as a biomarker of breast cancer.
Methods We enrolled 38 patients with breast cancer before surgery, two patients with noncancerous breast lesions, nine patients after surgery, 16 healthy participants, and 29 control women admitted to the hospital emergency ward and released without hospitalization. CFD levels were measured by a direct fluorescence assay.
Results Presurgery patients with cancer had elevated CFD levels (1,010 ± 642 ng/mL), which were higher than those measured in the healthy control group (395 ± 248 ng/mL, P < .001), the noncancer breast lesion group (386 ± 40 ng/mL), the nonhospitalized control group (492 ± 193 ng/mL, P < .001), and the postsurgery cancer group (398 ± 162 ng/mL, P < .01). The area under the receiver operating characteristic curve of the presurgery vs healthy patient group was 0.83. CFD levels correlated with tumor size (P = .03, ρ = 0.36), nodal involvement (P = .0003, ρ = 0.56), and TNM stage (P = .0002, ρ = 0.56). All patients with axillary node involvement had a CFD value greater than 600 ng/mL.
Conclusions CFD measured using a simple fluorometric assay has shown good correlation to stage and enhanced sensitivity to locally advanced disease. A large prospective study is warranted to evaluate if inclusion of this method as a decisive marker before mammography is advantageous.
Introduction
Breast cancer is now the most common cancer diagnosed in women, and it is the leading cause of deaths from cancer among women in the United States. The incidence is approximately 1.3 million new cases a year, and an estimated 458,000 deaths were reported in 2008. Since 1990, mortality from breast cancer in industrialized countries has been decreasing due to advances in adjuvant therapy and the increasing use of screening mammography (reviewed in Warner).
The current screening of breast cancer is based on clinical breast examination and mammography. Since breast cancer is known to have an asymptomatic phase, mammography is an important tool in the early diagnosis of breast cancer.
Mammography is considered highly sensitive and specific for women older than 50 years. However, 20% of patients diagnosed with breast cancer are younger than 50 years, and for this group of younger women, a sensitive screening alternative is presently not available. Mammography screening is recommended every 2 years, and between examinations, lack of specificity and sensitivity precludes the routine use of all available serum markers of breast cancer screening. Notwithstanding the wide use of mammography for the detection of breast cancer, it is worth noticing that serious questions have been raised about the value of screening mammography. A recent large randomized prospective study from Canada estimated that 22% of mammography screens overdiagnosed invasive breast cancers. These findings indicate the urgent need for new markers that will not only allow the screening of young women and their follow-up between examinations but also afford the critical discrimination between benign and malignant disease.
The presence of elevated levels of circulating cell-free DNA (CFD) in the blood of patients with cancer was initially demonstrated by Leon et al in 1977. The easy accessibility of plasma and serum DNA has made these noninvasive approaches an appealing method to detect and follow up patients with cancer. Many studies have confirmed the initial observation of Leon and colleagues and found elevated CFD levels in patients with malignancies (reviewed in Gormally et al), including patients with breast cancer. The elevation in circulating CFD levels originates from the release of DNA from cellular necrosis and apoptosis of tumor cells and probably from dying cells in the surrounding tissue affected by the tumor.
Although there is general agreement on the value of CFD measurement for patients with cancer, there is no standard method for its routine clinical use. The currently available research methods for CFD measurement are work-intensive and expensive, and they require DNA extraction and realtime polymerase chain reaction (PCR). Consequently, CFD measurements are not used for routine patient management.
We have developed a convenient and simple DNA assay applied directly to biological samples. This assay is based on the use of the nucleic acid fluorochrome SYBR Gold (Invitrogen, Paisley, England) stain, which does not require prior processing of samples (ie, DNA extraction and amplification). The assay is performed by adding diluted fluorochrome to the samples and measurement of fluorescence. Using this assay, we measured CFD levels and followed tumor growth and rejection in mice and in patients with colorectal cancer and found that CFD levels are prognostic for disease progression and death.
The aim of the current study was to evaluate the potential of this new method for the detection and prognosis of patients with breast cancer.
