Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-23
2001-11-13
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S378000, C536S024310
Reexamination Certificate
active
06316189
ABSTRACT:
FIELD OF THE INVENTION
This invention is related to the detection of chromosomal changes in memory epithelial cells obtained from nipple aspirate fluid as a screening tool for premalignant and malignant breast lesions.
BACKGROUND OF THE INVENTION
The increased availability of testing for breast cancer predisposing gene mutations (e.g. BRCA
1
and BRCA
2
) will soon result in a major increase in the number of young women identified as mutation carriers who require early and continuous surveillance. Means for early detection of chromosomal changes in women, especially those with family history of mutations, is needed. Mammography alone may not be sufficient for such evaluation. The effectiveness of mammography has not been established in women younger than 40 years of age. Younger women have more dense breast tissue, with reduced mammographic sensitivity. Moreover, tumor growth rates are often higher in younger women, thereby necessitating more frequent screening. However, carriers of some mutations (such as ataxia telangiectasia) may have increased sensitivity to radiation and could, conceivably, be harmed by frequent mammograms. Hence, new means of evaluation which avoid exposure to radiation would be particularly appropriate for use in younger women.
Nipple aspirate fluid (NAF) is secreted continuously by the non-lactating breast and, in 50% to 70% of premenopausal women, can be aspirated through duct openings in the nipple using a simple non-invasive pump. NAF is of interest because it has a relatively long retention time in the breast alveolar-ductal system, where it accumulates exfoliated mammary epithelial cells. Thus, cytogenetic examination of cells found in NAF would provide a “snapshot” of the micro-environment where breast cancer originates.
To date, classical cytologic assessment has been used to identify abnormalities in NAF-derived cells as an indicator of early progression toward breast cancer. However, NAF cytology alone is not sufficiently sensitive to identify the subgroup of women who are on a progression pathway that will lead to breast cancer. Atypical epithelial cells destined to progress to cancer have often accumulated a number of premalignant molecular changes. These changes are sufficiently subtle to require more sensitive, refined genetic analyses for their detection. Therefore, it is important to improve the sensitivity afforded by NAF cytology by examining these cells for chromosomal abnormalities.
SUMMARY OF THE INVENTION
This invention provides a method for studying chromosomal gains and losses in cells from nipple aspirate fluid comprising the steps of:
(1) aspirating fluid from breasts,
(2) placing the samples of fluid obtained in step 1 onto dishes containing conditioned medium composed of a mixture of (a) supernatant from immortalized mammary epithelial cells and (b) mammary epithelial growth medium,
(3) incubating the product of step 2 in a humidified incubator,
(4) replenishing the medium in the dishes prepared in step 2 at regular intervals to maintain cell growth,
(5) isolating the cells from the cultures,
(6) preparing DNA from these cells,
(7) amplifying the DNA,
(8) labeling an aliquot of the amplified DNA, and
(9) evaluating the labeled DNA for evidence of chromosomal gains or losses.
DESCRIPTION OF THE INVENTION
It is the purpose of this invention to provide a novel approach to early detection of premalignant and malignant changes. The use of this testing is particularly important to young women with inherited predisposing factors for breast tumors. The method of the invention uses mammary epithelial cells shed into nipple aspirate fluid to detect cytogenetic abnormalities associated with the early stages of progression toward breast cancer.
The method of the invention is exemplified using molecular cytogenetic methods of comparative genomic hybridization (CGH) to NAF-derived mammary epithelial cells to identify chromosomal gains and losses associated with early breast cancer. CGH permits the rapid screening of chromosomal imbalances within the test genomes without the need for metaphase preparations, only DNA from the test samples is need. While CGH has been exemplified, other methods such as chip technology may also be used in the practice of the invention.
The typical NAF sample from a woman with no breast abnormalities contains less than 10 ductal epithelial cells. Cells in the NAF are a heterogeneous cell population. The epithelial cellularity of NAF increases in the presence of benign or malignant breast abnormalities, as does the likelihood that the woman will secrete NAF. Furthermore, tumor cells are shed more readily than normal cells and probably have a selective growth advantage over normal cells. This could result in a preponderance of abnormal cells in the cultured sample. Chromosomal gains and losses, if present in these cells, can be detected by CGH.
Because NAF tends to contain small numbers of epithelial cells, it is necessary to culture live NAF-derived epithelial cells so that there will be sufficient cells to conduct the necessary cytogenetic studies. The instant method provides means of increasing the number of available cells from a sample to 250 to 500 cells. The DNA from these cells can then be isolated and amplified using means such as the universal DNA amplification procedure, degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The combination of culturing NAF-derived cells and use of universal DNA amplification followed by CGH or other means for detection of subtle genomic aberrations in those cells destined to progress to breast cancer can be a valuable diagnostic and screening tool.
REFERENCES:
Kelsey et al. Epidemiology and prevention of breast cancer. Annu. Rev. Public Health vol. 17 pp. 47-67, 1966.*
Gray et al. Molecular cytogenetics of human breast cancer. Cold Spring Symp. Quant. Biol. vol. 59 pages 645-652, 1994.*
Telenius et al. Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics vol. 13 pp. 718-725, 1992.*
Buehring et al. Growth rates of normal and abnormal human mammary epithelia in cell culture. Cancer Res. vol. 36 pp. 3742-3747, 1976.*
Bano et al. Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines. Biochemistry vol. 31 pp. 610-616, 1992.
Dickson Robert B.
Haddad Bassem R.
McCormack Stephen J.
Brusca John S.
Georgetown University School of Medicine
Teskin Robin L.
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