Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-19
2001-05-22
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007230, C435S330000, C530S387700, C530S388100, C530S388800
Reexamination Certificate
active
06235486
ABSTRACT:
BACKGROUND OF THE INVENTION
Breast cancer is a leading cause of mortality and morbidity among women. One of the priorities in breast cancer research is the discovery of new biochemical markers which can be used for diagnosis, prognosis and monitoring of breast cancer. The prognostic usefulness of these markers depends on the ability of the marker to distinguish between patients with breast cancer who require aggressive therapeutic treatment and patients who should be monitored. Because breast cancer is one of a few cancers that is dependent on steroid hormones and their receptors, analysis of estrogen receptor (ER) and progesterone receptor (PR) status is currently routinely performed as an aid in prognosis and selection of therapy. Other markers or indicators which are currently employed to diagnose and monitor breast cancer include: tumor size, age, aneuploidy, mitotic activity and Ki67 (Allred et al.,
J. Natl. Cancer Inst.,
85, 200-206 (1993)).
Mutation of the p53 tumor suppressor gene is one of the most commonly known genetic defects in human cancer, including breast cancer. Mutations in p53 result in the expression of a mutant protein which can accumulate to high concentrations. Overexpression of p53 protein is an independent predictor of early disease recurrence (Allred et al., supra). The accumulation of p53 protein has also been found to be an independent marker of shortened survival (Thor et al.,
J. Nat'l Cancer Inst.,
84, 845-855 (1992)). However, the majority of tumors that are estrogen and/or progesterone receptor-positive do not express mutant p53 protein.
Prostate cancer, like breast cancer, is dependent on steroid hormones. One of the hallmarks of prostate cancer is the appearance in serum, at elevated concentrations, of a 30-33 kD glycoprotein, prostate-specific antigen (PSA) (Oesterling,
J. Urol.,
145, 907-923 (1991)). PSA is a kallikrein-like serine protease that was thought to be exclusively produced by epithelial cells lining the acini and ducts of the prostate gland (Papsidero et al.,
J. Natl. Cancer Inst.,
66, 37-41 (1981); Lilja,
J. Clin. Invest.,
76, 1899-1903 (1985); Watt et al.,
Proc. Natl. Acad. Sci. USA,
83, 3166-3170 (1986)). Because of its tissue specificity, PSA has been widely used as a marker to diagnose and monitor prostate cancer (Stamey et al.,
N. Engl. J. Med.,
317, 909-916 (1987); Catalona et al.,
N. Engl. J. Med.,
324, 1156-1161 (1991)).
However, a number of studies have demonstrated the presence of PSA in non-prostate tissue. For example, Yu et al. (
Breast Cancer Res. Treat.,
32, 291-300 (1994)) reported that the steroid hormone receptor-positive breast carcinoma cell lines T47-D and MCF-7 can be stimulated by androgens, progestins, antiestrogens, mineralocorticoids and glucocorticoids to produce PSA. Diamandis et al. (
Breast Cancer Res. Treat.,
32, 301-310 (1994)) reported that 30% of female breast tumor cytosolic extracts contain PSA immunoreactivity. In addition, it is disclosed in Diamandis (WO 94/27152) that the presence of PSA in breast tumors is associated with tumors that express ER and/or PR. Thus, it has been speculated that PSA may be useful as a prognostic marker for breast cancer (Yu et al.,
Cancer Res.,
55, 2104-2110 (1995); Diamandis (WO 94/27152)).
Nevertheless, it is unclear whether PSA is correlated with ER and/or PR receptor status or has prognostic significance. In an analysis of a subset of breast tumors for PR and ER status, Yu et al. (
Clin. Biochem.,
27, 75 (1994)) disclose that immunoreactive PSA was only associated with PR, and no relationship was found between immunoreactive PSA and ER. In contrast, a 1995 report by Yu et al. (
Cancer Res.,
55, 2104 (1995)) found that PSA and ER were independent, although collaborative, markers for the prognosis of breast cancer. The authors also report that the presence or absence of PSA had no additional prognostic significance in steroid receptor-positive patients.
There is, therefore, a need for an inexpensive and simple prognostic and/or diagnostic marker for breast cancer that can function independently of, or in combination with, current employed markers.
SUMMARY OF THE INVENTION
The invention provides methods to determine the amount or presence of hK2 RNA or polypeptide in mammalian breast cells, e.g., breast tissue samples, or cells obtained from physiological fluids or tissue samples, e.g., blood or lymph node, which may comprise metastatic breast cancer cells. As described hereinbelow, hK2 is produced at a higher level relative to PSA by a breast cancer cell line, T47-D, after androgen stimulation. Moreover, T47-D cells produce significantly more hK2 than PSA (2-3 fold) when these cells are induced with mineralocorticoids, glucocorticoids or progestins. In contrast, estrogens failed to induce hK2. Therefore, the determination of the presence or amount of hK2 RNA or polypeptide may be useful in the diagnosis, treatment and/or monitoring of the progression or remission of breast cancer.
The invention thus provides a method for detecting hK2 nucleic acid in breast cells. The method comprises subjecting an amount of RNA obtained from a sample comprising breast cells to an amplification reaction so as to yield an amount of amplified nucleic acid, i.e., RNA or DNA. The amplified nucleic acid is then detected or determined. Methods to amplify nucleic acid molecules are well known to the art including, but not limited to, self-sustained sequence-specific replication (3SR) (Gebinoga et al.,
Eur. J. Biochem.,
235, 256 (1996); Fahy et al.,
PCR Methods Appl,
1, 25 (1991); Guatelli et al.,
Proc. Nat'l Acad. Sci. U.S.A.,
87, 1874 (1990)), nucleic acid sequence-based amplification (NASBA) (Compton,
Nature,
350, 91 (1991)), strand displacement amplification (SDA) (Walker et al.,
Proc. Nat'l Acad. Sci. U.S.A.,
89, 392 (1992); Walker et al.,
Nucl. Acid Res.,
20, 1691 (1992)), probe cyclization (Landgren,
Trends in Gen.,
2, 199 (1993)), or a Q beta replicase, Sp6, T7, or T3 RNA polymerase based amplification system. See, for example, U.S. Pat. Nos. 5,622,820, 5,629,153, 5,532,126, 5,573,914 and 5,514,545.
The invention also provides a method to detect hK2 cDNA. The method comprises contacting an amount of DNA obtained by reverse transcription (RT) of RNA from a sample comprising breast cells with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, at least one of which is an hK2-specific oligonucleotide, in an amplification reaction so as to yield an amount of amplified hK2 DNA. A preferred amplification reaction is a polymerase chain reaction (PCR). The presence of the amplified hK2 DNA is then detected. Preferably, the source of the sample to be tested is human tissue, more preferably, a human breast tissue biopsy sample, obtained from a male or female.
The invention further provides a method for detecting breast cancer in a human. The method comprises contacting an amount of DNA obtained by RT of RNA from a human physiological sample which comprises cells suspected of containing hK2 RNA, with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction, so as to yield an amount of amplified hK2 DNA. At least one oligonucleotide is an hK2-specific oligonucleotide. The presence or amount of the amplified hK2 DNA is detected or determined, and the presence or amount of the amplified hK2 DNA is then correlated to the presence or absence of breast cancer in said human.
Also provided is a diagnostic method for detecting hK2 RNA. The method comprises extracting RNA from a physiological sample obtained from a human. The extracted RNA is reverse transcribed to yield DNA. The DNA is contacted with an amount of at least two oligonucleotides effective to amplify the DNA to yield an amount amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide. The presence or amount of the amplified hK2 DNA is then detected or determined. The presence or amount of the amplified hK2 DNA is correlated to the presence or absence of
Klee George G.
Tindall Donald J.
Young Charles Y. F.
Mayo Foundation for Medical Education & Research
Schwegman Lundberg Woessner & Kluth P.A.
Stucker Jeffrey
Winkler Ulrike
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