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An Improved Breast Epithelial Sampling Method for Molecular Profiling and Biomarker Analysis in Women at Risk for Breast Cancer.

Danforth DN, Warner AC, Wangsa D, Ried T, Duelli D, Filie AC, Prindiville SA - Breast Cancer (Auckl) (2015)

Bottom Line: Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications.This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity.Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis.

View Article: PubMed Central - PubMed

Affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT

Background: There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity.

Methods: We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications.

Results: We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis.

Conclusion: An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.

No MeSH data available.


Related in: MedlinePlus

Cytopathologic illustration of epithelial cell content in ductal lavage samples. A ThinPrep Papanicolaou-stained slide of the cellular content for samples collected either using the original Cytyc microcatheter (A) or using the angiocatheter described in the present report (B). Panel A illustrates heterogeneity of the cell sample, showing both epithelial cells and foam cells with the original sampling method. The slide in (B) illustrates a more homogeneous population of ductal epithelial cells obtained with the current method. The slide in (C), same duct as in B, was immunorestained with cytokeratin antibodies to confirm the epithelial cell content.
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f1-bcbcr-9-2015-031: Cytopathologic illustration of epithelial cell content in ductal lavage samples. A ThinPrep Papanicolaou-stained slide of the cellular content for samples collected either using the original Cytyc microcatheter (A) or using the angiocatheter described in the present report (B). Panel A illustrates heterogeneity of the cell sample, showing both epithelial cells and foam cells with the original sampling method. The slide in (B) illustrates a more homogeneous population of ductal epithelial cells obtained with the current method. The slide in (C), same duct as in B, was immunorestained with cytokeratin antibodies to confirm the epithelial cell content.

Mentions: Each ductal lavage sample was examined for epithelial cell cytopathology and content. Representative slides illustrating epithelial cell content and confirmation by cytokeratin immunostaining are shown in Figure 1. Twelve subjects had ductal epithelial atypia on cytopathologic review, the majority of which were described as mildly atypical epithelial cells. Among these 12 cases of epithelial atypia, 3 occurred in high-risk subjects and the remainder occurred in women at normal risk for breast cancer. The findings for ductal cell yield are described in terms of the number of cells per sample and are summarized in Table 2. It can be seen that the ductal sampling method in an individual frequently provided multiple samples of high cellular content. For example, among the 48 subjects undergoing ductal lavage, 42 subjects produced one or more samples of ≥5,000 cells, and a median number of eight such samples (≥5,000 cells) per subject. Thirty-seven subjects (77%) had a median five samples of ≥10,000 epithelial cells per sample. When the samples were examined for the percentage of cells represented by epithelial cells (homogeneity of the sample), and using samples of ≥5,000 cells for calculations, it was found that a median eight samples per subject contained ≥80% epithelial cells, and a median six samples per subject contained ≥90% epithelial cells. Many samples comprised 99%–100% ductal epithelial cells (Fig. 1). We did not find any difference in cell yield between subjects with atypia compared to those without atypia.


An Improved Breast Epithelial Sampling Method for Molecular Profiling and Biomarker Analysis in Women at Risk for Breast Cancer.

Danforth DN, Warner AC, Wangsa D, Ried T, Duelli D, Filie AC, Prindiville SA - Breast Cancer (Auckl) (2015)

Cytopathologic illustration of epithelial cell content in ductal lavage samples. A ThinPrep Papanicolaou-stained slide of the cellular content for samples collected either using the original Cytyc microcatheter (A) or using the angiocatheter described in the present report (B). Panel A illustrates heterogeneity of the cell sample, showing both epithelial cells and foam cells with the original sampling method. The slide in (B) illustrates a more homogeneous population of ductal epithelial cells obtained with the current method. The slide in (C), same duct as in B, was immunorestained with cytokeratin antibodies to confirm the epithelial cell content.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4462519&req=5

f1-bcbcr-9-2015-031: Cytopathologic illustration of epithelial cell content in ductal lavage samples. A ThinPrep Papanicolaou-stained slide of the cellular content for samples collected either using the original Cytyc microcatheter (A) or using the angiocatheter described in the present report (B). Panel A illustrates heterogeneity of the cell sample, showing both epithelial cells and foam cells with the original sampling method. The slide in (B) illustrates a more homogeneous population of ductal epithelial cells obtained with the current method. The slide in (C), same duct as in B, was immunorestained with cytokeratin antibodies to confirm the epithelial cell content.
Mentions: Each ductal lavage sample was examined for epithelial cell cytopathology and content. Representative slides illustrating epithelial cell content and confirmation by cytokeratin immunostaining are shown in Figure 1. Twelve subjects had ductal epithelial atypia on cytopathologic review, the majority of which were described as mildly atypical epithelial cells. Among these 12 cases of epithelial atypia, 3 occurred in high-risk subjects and the remainder occurred in women at normal risk for breast cancer. The findings for ductal cell yield are described in terms of the number of cells per sample and are summarized in Table 2. It can be seen that the ductal sampling method in an individual frequently provided multiple samples of high cellular content. For example, among the 48 subjects undergoing ductal lavage, 42 subjects produced one or more samples of ≥5,000 cells, and a median number of eight such samples (≥5,000 cells) per subject. Thirty-seven subjects (77%) had a median five samples of ≥10,000 epithelial cells per sample. When the samples were examined for the percentage of cells represented by epithelial cells (homogeneity of the sample), and using samples of ≥5,000 cells for calculations, it was found that a median eight samples per subject contained ≥80% epithelial cells, and a median six samples per subject contained ≥90% epithelial cells. Many samples comprised 99%–100% ductal epithelial cells (Fig. 1). We did not find any difference in cell yield between subjects with atypia compared to those without atypia.

Bottom Line: Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications.This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity.Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis.

View Article: PubMed Central - PubMed

Affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT

Background: There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity.

Methods: We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications.

Results: We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis.

Conclusion: An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.

No MeSH data available.


Related in: MedlinePlus