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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

Array CGH of ductal lavage whole-genome-amplified DNA. This array is representative of DNA extracted from either the frozen pellet or the intact lavage suspension. Nonrandom gains are noted in chromosomes 6, 12, and 19.
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f3-bcbcr-9-2015-031: Array CGH of ductal lavage whole-genome-amplified DNA. This array is representative of DNA extracted from either the frozen pellet or the intact lavage suspension. Nonrandom gains are noted in chromosomes 6, 12, and 19.

Mentions: The quality of the DNA for subsequent analysis was further tested by whole-WGA and array CGH. Ten nanograms of genomic DNA was amplified, yielding microgram quantities of high-molecular-weight fragments for DNA extracted from both frozen pellets and the intact BDL suspension, and confirmed by agarose gel electrophoresis (data not shown). Hybridization of amplified DNA to Agilent human 4 × 44 K CGH arrays showed high-quality arrays for both types of sample preparation (frozen pellet or BDL suspension), with no differences noted (Fig. 3). Interestingly, occasional nonrandom gains were seen on chromosomes 6, 12, and 19 in all samples. The following genes are more highly gained in the respective segments: chromosome 6 – APOM; chromosome 12 – ARHGAP9, NDUFA4L2, CDK2, RHEBL1, TUBA1A, PRPH, LOC100335030, RACGAP1, LASS5, METTL7B; chromosome 19 – C19orf55, HIPK4, BCAM, RTN2, DHX34. Whether these gains represent artifacts of the amplification procedure or real genomic abnormalities of these normal breast epithelial samples needs to be determined.


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)

Array CGH of ductal lavage whole-genome-amplified DNA. This array is representative of DNA extracted from either the frozen pellet or the intact lavage suspension. Nonrandom gains are noted in chromosomes 6, 12, and 19.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-bcbcr-9-2015-031: Array CGH of ductal lavage whole-genome-amplified DNA. This array is representative of DNA extracted from either the frozen pellet or the intact lavage suspension. Nonrandom gains are noted in chromosomes 6, 12, and 19.
Mentions: The quality of the DNA for subsequent analysis was further tested by whole-WGA and array CGH. Ten nanograms of genomic DNA was amplified, yielding microgram quantities of high-molecular-weight fragments for DNA extracted from both frozen pellets and the intact BDL suspension, and confirmed by agarose gel electrophoresis (data not shown). Hybridization of amplified DNA to Agilent human 4 × 44 K CGH arrays showed high-quality arrays for both types of sample preparation (frozen pellet or BDL suspension), with no differences noted (Fig. 3). Interestingly, occasional nonrandom gains were seen on chromosomes 6, 12, and 19 in all samples. The following genes are more highly gained in the respective segments: chromosome 6 – APOM; chromosome 12 – ARHGAP9, NDUFA4L2, CDK2, RHEBL1, TUBA1A, PRPH, LOC100335030, RACGAP1, LASS5, METTL7B; chromosome 19 – C19orf55, HIPK4, BCAM, RTN2, DHX34. Whether these gains represent artifacts of the amplification procedure or real genomic abnormalities of these normal breast epithelial samples needs to be determined.

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