Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-06
2001-04-17
Wortman, Donna C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S324000, C530S326000, C530S327000, C530S328000, C530S329000, C530S387700, C530S387900
Reexamination Certificate
active
06218131
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to materials and methods for the detection of breast cancer, including cellular markers indicative of the likelihood of the presence of breast cancer.
BACKGROUND OF THE INVENTION
Breast cancer is a leading cause of death in women. While the pathogenesis of breast cancer is unclear, transformation of normal breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under 30. Miki, et al.,
Science
, 266: 66-71 (1994). However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease. Regardless of its origin, breast cancer morbidity increases significantly if a lesion is not detected early in its progression. Thus, considerable effort has focused on the elucidation of early cellular events surrounding transformation in breast tissue. Such effort has led to the identification of several potential breast cancer markers. For example, alleles of the BRCA1 and BRCA2 genes have been linked to hereditary and early-onset breast cancer. Wooster, et al.,
Science
, 265: 2088-2090 (1994). The wild-type BRCA1 allele encodes a tumor supressor protein. Deletions and/or other alterations in that allele have been linked to transformation of breast epithelium. Accordingly, detection of mutated BRCA1 alleles or their gene products has been proposed as a means for detecting breast, as well as ovarian, cancers. Miki, et al., supra. However, BRCA1 is limited as a cancer marker because BRCA1 mutations fail to account for the majority of breast cancers. Ford, et al.,
British J. Cancer
, 72: 805-812 (1995). Similarly, the BRCA2 gene, which has been linked to forms of hereditary breast cancer, accounts for only a small portion of total breast cancer cases. Ford, et al., supra.
Several other genes have been linked to breast cancer and may serve as markers for the disease, either directly or via their gene products. Such potential markers include the TP53 gene and its gene product, the p53 tumor supressor protein. Malkin, et al.,
Science
, 250: 1233-1238 (1990). The loss of heterozygosity in genes such as the ataxia telangiectasia gene has also been linked to a high risk of developing breast cancer. Swift, et al.,
N. Engl. J. Med
., 325: 1831-1836 (1991). A problem associated with many of the markers proposed to date is that the oncogenic phenotype is often the result of a gene deletion, thus requiring detection of the absence of the wild-type form as a predictor of transformation.
Of interest to the present invention are reports that the protein content of the nuclear matrix in breast epithelia may provide a marker of cellular growth and gene expression in those cells. Khanuja, et al.,
Cancer Res
., 53: 3394-3398 (1993). The nuclear matrix forms the superstructure of the cell nucleus and comprises multiple protein components that are not fully characterized. The nuclear matrix also provides the structural and functional organization of DNA. For example, the nuclear matrix allows DNA to form loop domains. Portions of DNA in such loop domains have been identified as regions comprising actively-transcribing genes. Ciejek, et al.,
Nature
, 306: 607-609 (1982). Moreover, the organization of the nuclear matrix appears to be tissue-specific and has been associated with so-called transformation proteins in cancer cells. Getzenberg, et al.,
Cancer Res
., 51: 6514-6520 (1991); Stuurman, et al.,
J. Biol. Chem
., 265: 5460-5465 (1990).
Proteins and steroid hormones thought to be involved in transformation are associated with the nuclear matrix in certain cancer cells. Getzenberg, et al.,
Endocrinol. Rev
., 11: 399-417 (1990). It has been suggested that changes in the composition or organization of nuclear matrix proteins may be useful as markers of growth and gene expression in breast tissue. Khanuja, et al.,
Cancer Res
., 53: 3394-3398 (1994). However, Khanuja did not identify any specific proteins for use as cancer markers.
There is, therefore, a need in the art for specific, reliable markers that are differentially expressed in normal and transformed breast tissue and that may be useful in the diagnosis of breast cancer or in the prediction of its onset. Such markers and methods for their use are provided herein.
SUMMARY OF THE INVENTION
The invention provides materials and methods for diagnosis and detection of breast cancer in tissue or in body fluid. In a preferred embodiment, methods according to the invention comprise the step of detecting in a sample of tissue or body fluid the presence of a protein that is not normally expressed in non-transformed (i.e., noncancerous) breast cells. Such proteins are typically found in the nuclear matrix fraction of cells or cellular material isolated according to the method of Fey, et al.
Proc. Nat'l. Acad. Sci
. (USA), 85: 121-125 (1988), incorporated by reference herein. Accordingly, such proteins are alternatively referred to herein as breast cancer-associated proteins or breast cancer-associated nuclear matrix proteins. It is understood that, for purposes of the present invention, a breast cancer-associated protein, including a nuclear matrix protein, is one that is detectable in breast cancer cells and not detectable in non-cancerous cells and which can be isolated as described herein.
Methods of the invention may be performed on any relevant tissue or body fluid sample. In preferred embodiments, methods of the invention are carried out in breast tissue and preferably breast biopsy tissue. However, inventive methods are also useful in assays for metastasized breast cancer cells in other tissue or body fluid samples. Methods for detecting breast cancer-associated proteins in breast tissue may comprise exposing such tissue to an antibody directed against a target breast cancer-associated protein. The antibody may be polyclonal or monoclonal and may be detectably labeled for identification of antibody.
A detecting step according to the invention may comprise amplifying nucleic acid encoding a target breast cancer-associated protein using a polymerase chain reaction or a reverse-transcriptase polymerase chain reaction. Detection of products of the polymerase chain reaction may be accomplished using known techniques, including hybridization with nucleic acid probes complementary to the amplified sequence. A detecting step according to the present invention may also comprise using nucleic acid probes complementary to at least a portion of a DNA or RNA encoding a breast cancer-associated protein.
The present invention also provides proteins and protein fragments that are characteristic of breast cancer cells. Such proteins and protein fragments are useful in the detection and diagnosis of breast cancer as, for example, in the production of antibodies. The invention also provides nucleic acids encoding breast cancer-associated proteins. The nucleic acids themselves are contemplated as markers and may be detected in order to establish the presence of breast cancer or a predisposition therefor.
In a preferred embodiment, methods of the invention comprise the step of detecting these proteins and/or nucleotides. Specifically, methods of the invention comprise detecting a breast cancer-associated protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20; and/or a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.
Breast cancer-associated proteins in a tissue or body fluid sample may be detected using any assay method available in the art. In one embodiment, the protein may be reacted with a binding moiety, such as an antibody, capable of specifically binding the protein being detected. Binding moieties, such as antibodies, may be designed using methods available in the art so that they interact specifically with the protein being detected. Optionally, a labeled binding moiety may be utilized. In such an embodiment, the sample is reacted with a labeled binding moiety capable of specifically binding the
Keesee Susan K.
Obar Robert
Wu Ying-Jye
Brumback Brenda G.
Matritech, Inc.
Testa Hurwitz & Thibeault LLP
Wortman Donna C.
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