Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-31
2003-04-08
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06544742
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a composition comprising a plurality of polynucleotide probes which may be used in detecting expression of genes modulated in response to EGF, and which are associated with breast cancer.
BACKGROUND OF THE INVENTION
Intercellular communication is essential for the development and survival of multicellular organisms. Communication is achieved through the secretion of proteins by signaling cells and the internalization of these proteins by target cells. Growth factors are secreted proteins that mediate communication between signaling and target cells. The secreted growth factors bind to specific receptors on the surfaces of target cells, and bound receptors trigger second messenger signal transduction pathways. These signal transduction pathways elicit specific cellular responses in the target cells. Such responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
Epidermal growth factor (EGF) is a member of a broad class of polypeptide growth factors that generally act as mitogens in diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues. In addition, EGF produces non-mitogenic effects in certain tissues. The EGF receptor (EGFR), and its stimulation by EGF, has been linked with a number of cell proliferative disorders or malignancies. These include skin hyperplasia, erythroblastosis, and fibrosarcoma in animals; and in humans, benign hyperplasia of the skin, mammary carcinoma, glioblastoma, and hepatic carcinoma. Other epithelial carcinomas associated with EGFR activity include prostatic hyperplasia/cancer, renal carcinoma, bladder cancer, laryngeal cancer, esophageal tumors, stomach cancer, colon carcinoma, ovarian adenomas, and lung cancer (Khazaie, K. et al. (1993) Cancer and Metastasis Rev. 12:255-274).
The relationship of EGFR expression to human mammary carcinoma has been particularly well studied. (See Khazaie et al., supra, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor (ER), has been a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has pleiotropic effects on cell motility, chemotaxis, secretion and differentiation; cell functions related to metastatic potential. For example, EGF has been found to influence the expression and organization of integrins, a family of receptors known to function in cell attachment to the extracelluar matrix during metastasis (Nicolson, G. L. (1984) Expl. Cell Res. 150:3-22; Schirrmacher, V (1985) Adv. Cancer Res. 43:1-73). EGFR may influence cell-cell adhesion by affecting changes in the phosphorylation of certain proteins involved in the process, such as &bgr;-atenin, fodrin, spectrin, and tubulin (Khazaie et al., supra). EGF has also been shown to affect the production and release of various proteinases involved in cell invasion of the extracelluar matrix, such as metalloproteinases, aminopeptidases, serine proteases, cysteine proteases and aspartic proteinases, as well as proteinase inhibitors such as plasminogen activator inhibitor (PAI-1) and tissue inhibitors of metalloproteases (TIMP).
In addition to the various proteins indicated above that are affected by EGF activity, the EGF signal transduction pathway itself involves the recruitment and activation of a variety of molecules including phospholipase C, phosphoinositol-3 kinase, MAP kinase, raf kinase, and a GTPase-activating protein (GAP). The expression of these and other molecules effected by EGF activity may be useful for the prediction or monitoring of cell proliferative disorders, pre-malignant conditions, or the presence and progression of malignant diseases in which EGF participates.
Array technology can provide a simple way to explore the expression of a single polymorphic gene or a large number of related or unrelated genes. When the expression of a single gene is explored, arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer. The array has over tens of thousands of DNA probes to analyze more than 400 distinct mutations of p53.
DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes. There is a growing awareness that gene expression is affected in a global fashion. A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes. In some cases the interactions may be expected, such as where the genes are part of the same signaling pathway. In other cases, the interactions may be totally unexpected, such as when the genes participate in separate signaling pathways. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.
The potential application of gene expression profiling to breast cancer is particularly relevant to improving diagnosis and prognosis of this disease. The mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (K. Gish (1999) AWIS Magazine, 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are, however, lacking in sensitivity and specificity, and efforts are underway in other laboratories to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Gish, supra).
It would be advantageous to prepare DNA-based arrays that can be used for monitoring expression of a large number of genes associated with cell proliferative disorders and with pre-malignant and malignant conditions. The present invention provides for a composition comprising a plurality of polynucleotide probes for use in detecting changes in expression of a large number of genes encoding proteins associated with EGFR expression and activity. Such a microarray can be employed for diagnosis and monitoring of the treatment of any disease or precondition where EGFR activation is involved, in particular, breast cancer.
SUMMARY
The present invention provides a combination comprising a plurality of cDNAs, wherein each of the cDNAs comprises at least a fragment of a polynucleotide sequence or a complement thereof whose expression is modulated by EGF and is associated with breast cancer and which are selected from SEQ ID NOs: 1-16 as presented in the Sequence Listing. In one aspect, the combination is immobilized on a substrate.
The invention also provides a high throughput method to detect differential expression of one or more of the cDNAs of the combination. The method comprises hybridizing the substrate comprising the combination with the nucleic acids of a sample, thereby forming one or more hybridization complexes, detecting the hybridization complexes, and comparing the hybridization complexes with those of a standard, wherein differences in the size and signal intensity of each hybridization complex indicates differential expression of nucleic acids in the sample.
The invention further provides a high throughput method of screening a library or a plurality of molecules or compounds to identify a ligand. The method comprises combining the substrate comprising the combination with a library or a plurality of molecules or compounds
Faris Mary
Streeter David G.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Jones W. Gary
Souaya Jehanne
LandOfFree
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