Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-14
2001-10-16
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007200, C435S023000, C536S023500, C536S024310, C530S350000
Reexamination Certificate
active
06303318
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the fields of molecular biology and medicine. More specifically, the present invention is in the field of ovarian and other cancer diagnosis.
BACKGROUND OF THE INVENTION
To date, ovarian cancer remains the number one killer of women with gynecologic malignant hyperplasia. Approximately 75% of women diagnosed with such cancers are already at the high-stage (III and IV) of the disease at their initial diagnosis. During the past 20 years, neither diagnosis nor five year survival have greatly improved for these patients. This is substantially due to the high percentage of high-stage initial detections of the disease. Therefore, the challenge remains to develop new markers to improve early diagnosis, and reduce the percentage of high-stage initial diagnoses.
A good tumor marker useful as an indicator of early disease is needed. Extra-cellular proteases have already been implicated in the growth, spread and metastatic progression of many cancers, thereby implying that some extracellular proteases may be candidates for marker of neoplastic development. This is in part due to the ability of malignant cells not only to grow in situ, but to dissociate from the primary tumor and to invade new surfaces. The ability to disengage from one tissue and re-engage the surface of another tissue is what provides for the morbidity and mortality associated with this disease.
In order for malignant cells to grow, spread or metastasize, they must have the capacity to invade local host tissue, dissociate or shed from the primary tumor, and for metastasis to occur, enter and survive in the bloodstream, implant by invasion into the surface of the target organ and establish an environment conducive for new colony growth (including the induction of angiogenic and growth factors). During this progression, natural tissue barriers have to be degraded including basement membranes and connective tissue. These barriers include collagen, laminin, proteoglycans and extracellular matrix glycoproteins including fibronectin.
Degradation of these natural barriers, both surrounding the primary tumor and at sites of metasttic invasion is believed to be brought about by the action of a matrix of extracellular protease. Proteases have been classified into four families: serine proteases, metallo-proteases, aspartic proteases and cysteine proteases. Many proteases have been shown to be involved in the human disease process and these enzymes are targets for the development of inhibitors as new therapeutic agents.
Certain individual proteases have already been shown to be induced and over expressed in a diverse group of cancers, and as such, are potential candidates for markers useful for early diagnosis and possibly therapeutic intervention. A group of examples are listed below. The list of enzymes encompasses members of the metallo-proteases, serine proteases, and cysteine proteases as shown in Table 1.
TABLE 1
Protease Expression in Various Cancers
Gastric
Brain
Breast
Ovarian
Serine Proteases
uPA
uPA
NES-1
NES-1
PAI-1
PAI-1
uPA
uPA
tPA
PAI-2
Cysteine Proteases
Cathepsin B
Cathepsin L
Cathepsin B
Cathepsin B
Cathepsin L
Cathepsin L
Cathepsin L
Metallo-proteases
Matrilysin*
Matrilysin
Stromelysin-3
MMP-2
Collagenase*
Stromelysin
MMP-8
Stromelysin-1* Gelatinase BMMP-9
Gelatinase A
uPA-Urokinase-type plasminogen activator, tPA - Tissue-type plasminogen activator, PAI-I - Plasminogen
# activator 0 inhibitors, PAI-2 - Plasminogen activator inhibitors, NES-1 - Normal epithelial cell-specific-1,
# MMP - Matrix P metallo-protease.
*These metallo-proteases are over expressed in gastrointestinal ulcers.
Significantly there is a good body of evidence supporting the down regulation or inhibition of individual proteases and reduction in invasive capacity or malignancy. In work by Clark et al. inhibition of in vitro growth of human small cell lung cancer was demonstrated using a general serine protease inhibitor. More recently, Torres-Rosedo et al.,
Proc. Natl. Acad. Sci. USA,
90, 7181-7185 (1993). demonstrated an inhibition of tumor cell growth of hepatoma cells using specific antisense inhibitors for the serine protease hepsin gene. Metastatic potential has also been shown to be reduced in a mouse model with melanoma cells by using a synthetic inhibitor (batimastat) of metallo-protease. Powell, et al.
Cancer Research,
53, 417-422 (1993), presented evidence to confirm that the expression of extracellular proteases in relatively non-invasive tumor cells enhances their malignant progression using a tumor-genic but non-metastatic prostate cell line. Specifically, they demonstrated enhanced metastasis after introducing and expressing the PUMP-1 metallo-protease gene. There is also a body of data to support the notion that expression of cell surface proteases on relatively non-metastatic cell types increases the invasive potential of such cells.
SUMMARY OF THE INVENTION
This invention detects the presence of cancers, especially ovarian cancer, by screening for a plurality of mRNA markers in tissue, which markers are indicative of proteases specifically associated with the surface of 80 percent of ovarian tumors, and other tumors. Specific combinations of proteases are characteristic of particular tumor types as is illustrated below. These proteases are considered to be an integral part of tumor growth and metastasis and therefore, markers indicative of their presence or absenceare useful for the diagnosis of cancer. The invention provides a method for detecting malignant hyperplasia in a biological sample comprising the steps of isolating the proteases or protease mRNA present in the tissue sample;
detecting and identifying specific proteases present in the tissue sample from the group of proteases consisting of Stratum Corneum Chymotrytic Enzyme (SCCE), TADG12, TADG13, TADG14, Hepsin, Punp-1 and Protease M. Preferably further comprising the step of comparing the specific proteases detected to reference information and providing a diagnoses based in part on the identification of specific proteases associated with the biological sample. In a preferred mode the invention allows identification of specific tumors based on the expression of particular proteases, or the absence of specific proteases. Alternatively, the method may comprise the step of comparing the specific proteases detected to reference information and providing a treatment based in part on the identification of specific proteases associated with the biological sample. In a preferred mode, the invention allows selection of a treatment based on the expression of particular proteases or the absence of particular proteases. A protease is identified by isolation of and amplification of protease mRNA. Alternatively, a protease is isolated by an antibody. The biological sample may be tissue, or preferably a bodily fluid or more preferably blood or a blood component.
The invention further provides a method for detecting ovarian malignant hyperplasia in a biological sample comprising the steps of:
isolating the proteases or protease mRNA present in the tissue sample;
detecting and identifying specific proteases present in the tissue sample from the group of proteases consisting of Hepsin, Protease M, Complement factor B, SCCE, Serine proteases indicated at Lanes 2 and 4,
FIG. 1
TADG12, TADG13, TADG14, Cysteine protease Cathepsin L, and metalo-protease Pump-1. Preferably this method further comprising the step of comparing the specific proteases detected to reference information and providing a diagnoses based in part on the identification of specific proteases associated with the biological sample. Alternatively, the method may comprise the step of comparing the specific proteases detected to reference information and providing a treatment based in part on the identification of specific proteases associated with the biological sample. A protease is identified by isolation of mRNA, conversion of the isolated mRNA to cDNA and amplification of the converted protease cDNA. Alternatively, a protease is isolated or detected by an
Adler Benjamin Aaron
Myers Carla J.
The Board of Trustees of the University of Arkansas
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