Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-04-11
2002-11-12
Ungar, Susan (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S009000, C536S024300, C536S024310, C536S024330, C536S025300
Reexamination Certificate
active
06479263
ABSTRACT:
BACKGROUND OF THE INVENTION
The glandular kallikreins are a subgroup of serine proteases which are involved in the post-translational processing of specific polypeptide precursors to their biologically active forms. In humans, three members of this family have been identified, and some of their properties characterized (Clements,
Endoc. Rev
., 10, 343 (1989); Clements,
Mol. Cell Endo
., 99, 1 (1994); Jones et al.,
Acta Endoc
., 127, 481 (1992)). The hKLK1 gene encodes the tissue kallikrein protein, hK1, the hKLK2 gene encodes the prostate-specific glandular kallikrein protein, hK2, and the hKLK3 gene encodes the prostate-specific antigen protein, hK3 (PSA). Northern blot analysis of mRNA shows that both hK2 and PSA are expressed mainly in the human prostate, while expression of hK1 is found in the pancreas, submandibular gland, kidney, and other nonprostate tissues (Chapdelaine et al.,
FEBS Lett
., 236, 205 (1988); Young et al.,
Biochem
. 31, 818 (1992)).
The nucleotide sequence homology between the exons of hKLK2 and hKLK3 is 80%, whereas the nucleotide sequence homology between the exons of hKLK2 and hKLK1 is 65%. The deduced amino acid sequence homology of hK2 to PSA is 78%, whereas the deduced amino acid sequence homology of hK2 to hK1 is 57%. Moreover, the deduced amino acid sequence of hK2 suggests that hK2 may be a trypsin-like protease, whereas PSA is a chymotrypsin-like protease.
PSA levels are widely used as a prognostic indicator of prostate carcinoma. However, since the concentration of PSA in serum is elevated in patients with either prostatic cancer (pCa) or benign prostatic hyperplasia (BPH), the detection of elevated levels of PSA does not distinguish between these diseases. Moreover, the high degree of homology of hK2 to PSA raises some question as to the specificity of antibodies currently used to detect the levels of PSA. If the levels of circulating hK2 are unrelated to pCa or BPH, then antibodies raised to preparations of PSA which are contaminated with hK2, or to regions of PSA with homology to hK2, can result in false positive results.
Although it is now generally accepted that serum PSA testing, combined with the digital rectal exam (DRE), is the most effective means to detect clinically significant and organ-confined prostate cancer, combinations of PSA, DRE and ultrasonic prostate examination can detect only some prostate tumors. For example, up to 40% of surgically treated patients with prostate cancer are subsequently found to be clinically understaged. Moreover, the actual incidence of histological cancers based on autopsy data relative to the incidence of clinically significant prostate cancer is high. Furthermore, approximately 30% of patients with alleged localized prostate cancer may have occult (distant) metastatic disease (Moreno et al.,
Cancer Res
., 52, 6110 (1992)). Of these patients, 80% experience relapse biochemically, i.e., elevated PSA levels, or by the recurrence of local, or occurrence of frank systemic, disease, after therapy.
Operative therapy is not the appropriate treatment modality for patients having established metastasis. Screening modalities to assess early metastases often fail to identify a significant subset of patients with locally invasive disease involving penetration of the prostate capsule or seminal vesicle. While immunohistochemical techniques have been employed to identify micrometastatic, or circulating, prostate tumor cells when no obvious metastatic deposit was evidenced by conventional means, immunohistochemical methods are laborious and lack the sensitivity needed for the early detection of metastatic or locally invasive prostate cancer.
There is, therefore, a need for early detection of prostate cancer cells with metastatic potential. Moreover, there is a need to accurately stage prostate cancer prior to subjecting a patient to invasive procedures. In particular, there is a need for a marker for prostate cancer that can function independently of, or in combination with, PSA.
SUMMARY OF THE INVENTION
The invention provides a diagnostic method for detecting hK2 DNA wherein the presence of prostate cancer cells in a physiological sample can be correlated to the detection of hK2 RNA in the sample. Because expression of hK2 is prostate tissue specific, hK2 RNA should theoretically not be detectable in cells present in bodily fluids or non-prostate tissue if there is no locally invasive or metastatic disease, or if all prostate tissue (benign and malignant) has been removed or destroyed. The method comprises contacting an amount of DNA obtained by reverse transcription (RT) of RNA from a human physiological sample with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, at least one of which 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 conditions are effective to amplify the amount of DNA obtained by reverse transcription of RNA from at least one cell containing hK2 in a sample which comprises at least about 10
7
to about 10
9
cells. As described hereinbelow, the presence of amplified hK2 DNA in blood cells, after RT-PCR, is correlated with prostate cancer, i.e., sixty-seven percent (67%) of the prostate cancer patients expressed hK2, 17% expressed PSA, and 17% expressed both hK2 and PSA. Preferably, the source of the sample to be tested is human tissue, e.g., prostate, prostate capsule, seminal vesicle, bone marrow or lymph node. Another preferred source of the sample to be tested is a human physiological fluid which comprises cells, e.g., blood, serum, or seminal fluid.
As used herein, “amplified hK2 DNA” is defined to mean hK2 DNA in a sample, which was subjected to an amplification reaction, that is present in an amount that is greater than, i.e., 10, preferably 10
4
, and more preferably 10
6
, times greater than, the amount of hK2 DNA which was present in the sample prior to amplification.
As used herein, the term “hK2-specific oligonucleotide” or “hK2-specific primer” means a DNA sequence that has at least about 80%, more preferably at least about 90%, and more preferably at least about 95%, sequence identity or homology to SEQ ID NO:4 in regions of SEQ ID NO:4 that are divergent from nucleotide sequences which encode hK3 (SEQ ID NO:23). An oligonucleotide or primer of the invention has at least about 7-50, preferably at least about 10-40, and more preferably at least about 15-35, nucleotides. Preferably, the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3′ of the oligonucleotide primer which have at least about 80%, more preferably at least about 85%, and more preferably at least about 90%, identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The oligonucleotides of the invention may also include sequences which are unrelated to hK2 nucleic acid sequences, e.g., they may encode restriction endonuclease recognition sequences. A preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:14. Another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:17. Yet another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:18.
A preferred diagnostic method of the invention combines RT-PCR detection of hK2 transcripts with RT-PCR detection of transcripts of other gene products associated with prostate cancer. Combined detection of two or more gene products may provide greater diagnostic certainty or yield more informative staging information. Combined detection may also be helpful in differentiating those cells with aggressive growth potential from those that are more indolent. In a particularly preferred embodiment of the method provided by the invention, RT-PCR detection of hK2 RNA is combined with RT-PCR detection of PSA RNA.
The invention further provides a diagnostic method for detecting hK2 RNA. The method comprises extracting RNA from a physiological sam
Kumar Abhay
Rittenhouse Harry G.
Saedi Mohammad Saeed
Slawin Kevin M.
Tindall Donald J.
Baylor College of Medicine
Davis Minh-Tam
Schwegman - Lundberg Woessner - Kluth
Ungar Susan
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