Patent Grant
8703127
This application incorporates by reference in its entirety the Sequence Listing contained in the accompanying file, named “1003CON_seqlist.txt,” the size of which is 30 KB, and which was created on Jan. 13, 2012.
The present disclosure relates to methods and compositions for diagnosing, and monitoring the progress of, prostate cancer.
The use of prostate specific antigen (PSA) as a diagnostic biomarker for prostate cancer was approved by the US Federal Drug Agency in 1994. In the nearly two decades since this approval, the PSA test has remained the primary tool for use in prostate cancer diagnosis, in monitoring for recurrence of prostate cancer, and in following the efficacy of treatments. However the PSA test has multiple shortcomings and, despite its widespread use, has resulted in only small changes in the death rate from advanced prostate cancers. To reduce the death rate and the negative impacts on quality of life caused by prostate cancer, new tools are required not only for more accurate primary diagnosis, but also for assessing the risk of spread of primary prostate cancers, and for monitoring responses to therapeutic interventions.
Today, a blood serum level of around 4 ng per ml of PSA is considered indicative of prostate cancer, while a PSA level of 10 ng per ml or higher is considered highly suggestive of prostate cancer.
The PSA blood test is not used in isolation when checking for prostate cancer; a digital rectal examination (DRE) is usually also performed. If the results of the PSA test or the DRE are abnormal, a biopsy is generally performed in which small samples of tissue are removed from the prostate and examined. If the results are positive for prostate cancer, further tests may be needed to determine the stage of progression of the cancer, such as a bone scan, a computed tomography (CT) scan or a pelvic lymph node dissection.
While the PSA test has a good sensitivity (80%), it suffers from a false positive rate that approaches 75%. For example, it has been estimated that for PSA values of 4-10 ng/ml, only one true diagnosis of prostate cancer was found in approximately four biopsies performed (Catalona et al. (1994) J. Urol. 151(5):1283-90). Tests that measure the ratio of free to total (i.e., free plus bound) PSA do not have significantly greater specificity or sensitivity than the standard PSA test.
Higher PSA levels often lead to biopsies to determine the presence or absence of cancer cells in the prostate, and may lead to the surgical removal of the localized prostate gland. While surgery removes the localized cancer and often improves prostate cancer-specific mortality, it also masks the fact that many patients with prostate cancer, even in the absence of surgery, do not experience disease progression to metastasis or death.
Currently, the established prognostic factors of histological grade and cancer stage from biopsy results, and prostate-specific antigen level in blood at diagnosis are insufficient to separate prostate cancer patients who are at high risk for cancer progression from those who are likely to die of another cause.
Once virulent forms of prostate cancer have been diagnosed, control strategies may involve surgery to remove the prostate gland if identified before metastasis, radiation to destroy cancer cells within the prostate, and drug-based testosterone repression, generally referred to as androgen depletion therapy. These various treatments may bring about cures in some instances, or slow the time to death. However, for those with the most virulent forms of prostate cancer, the cancer will usually recur after surgery or radiation therapy and progress to resistance to androgen depletion therapy, with death a frequent outcome.
Early detection of virulent forms of prostate cancer is critical but the conclusion of specialist physicians is that the PSA test alone is inadequate for distinguishing patients whose cancers will become virulent and progress to threaten life expectancy from those with indolent cancers.
The following are some key reasons why the PSA test does not meet the needs of men's health:
i) The Type of Cancer
There are at least two basic cell types involved in prostate cancer. Adenocarcinoma is a cancer of epithelial cells in the prostate gland and accounts for approximately 95% of prostate cancers. Neuroendocrine cancers may arise from cells of the endocrine (hormonal) and nervous systems of the prostate gland and account for approximately 5% of prostate cancers. Neuroendocrine cells have common features such as special secretory granules, produce biogenic amines and polypeptide hormones, and are most common in the intestine, lung, salivary gland, pituitary gland, pancreas, liver, breast and prostate. Neuroendocrine cells co-proliferate with malignant adenocarcinomas and secrete factors which appear to stimulate adenocarcinoma cell growth. Neuroendocrine cancers are rarer, and are considered non-PSA secreting and androgen-independent for their growth.
ii) Asymptomatic Men
Some 15 to 17% of men with prostate cancer have cancers that grow but do not produce increasing or high blood levels of PSA. In these patients, who are termed asymptomatic, the PSA test often returns false negative test results as the cancer grows.
iii) BPH, Prostatitis and PIN
Benign prostate hypertrophy (BPH), a non-malignant growth of epithelial cells, and prostatitis are diseases of the prostate that are usually caused by an infection of the prostate gland. Both BPH and prostatitis are common in men over 50 and can result in increased PSA levels. Incidence rates increase from 3 cases per 1000 man-years at age 45-49 years, to 38 cases per 1000 man-years by the age of 75-79 years. Whereas the prevalence rate is 2.7% for men aged 45-49, it increases to at least 24% by the age of 80 years. While prostate cancer results from the deregulated proliferation of epithelial cells, BPH commonly results from proliferation of normal epithelial cells and frequently does not lead to malignancy (Ziada et al. (1999) Urology 53(3 Suppl 3D):1-6). Prostatitis in males is frequently attributed to an infection in prostate tissue including infection by E. coli, Klebsiella sp., Serratia and/or Pseudomonas sp.
Another condition, known as prostate intraepithelial neoplasia (PIN), may precede prostate cancer by five to ten years. Currently there are no specific diagnostic tests for PIN, although the ability to detect and monitor this potentially pre-cancerous condition would contribute to early detection and enhanced survival rates for prostate cancer.
iv) The Phenotype of the Prostate Cancer
The phenotype of prostate cancer varies from one patient to another. More specifically, in different individuals prostate cancers display heterogeneous cellular morphologies, growth rates, responsiveness to androgens and pharmacological blocking agents for androgens, and varying metastatic potential. Each prostate cancer has its own unique progression involving multiple steps, including progression from localized carcinoma to invasive carcinoma to metastasis. The progression of prostate cancer likely proceeds, as seen for other cancers, via events that include the loss of function of cell regulators such as cancer suppressors, cell cycle and apoptosis regulators, proteins involved in metabolism and stress response, and metastasis related molecules (Abate-Shen et al. (2000) Polypeptides Dev. 14(19):2410-34; Ciocca et al. (2005) Cell Stress Chaperones 10(2):86-103).
There is unlikely to be one diagnostic test that detects all forms of primary prostate cancer, or one prognostic test for all routes to metastasis. Further tests are therefore needed before prostate cancer can be diagnosed in its different forms.
At present health authorities, do not universally recommend widespread screening for prostate cancer with the PSA test. There are concerns that many men may be diagnosed and treated unnecessarily as a result of being screened, at high cost to health systems as well as risking the patient's quality of life, such as through incontinence or impotence. Despite these concerns, prostate cancer is the most prevalent form of cancer and the second most common cause of cancer death in New Zealand, Australian and North American males (Jemal et al. (2007) C A Cancer J. Clin. 57(1):43-66). In reality, at least some of the men incubating life threatening forms of prostate cancer are being missed until their cancer is too advanced, due to the economic costs of national screening, the need to avoid unnecessary over-treatment, and/or the presence of progressive cancers producing only low or background levels of PSA. The need for a better diagnostic test could not be clearer.
An important clinical question is how aggressively to treat patients with localized prostate cancer. Treatment options for more aggressive cancers are invasive and include radical prostatectomy and/or radiation therapy. Androgen-depletion therapy, for example using gonadotropin-releasing hormone agonists (e.g., leuprolide, goserelin, etc.), is designed to reduce the amount of testosterone that enters the prostate gland and is used in patients with metastatic disease, some patients who have a rising PSA and choose not to have surgery or radiation, and some patients with a rising PSA after surgery or radiation. Treatment options usually depend on the stage of the prostate cancer. Men with a 10-year life expectancy or less, who have a low Gleason score from a biopsy and whose cancer has not spread beyond the prostate are often not treated. Younger men with a low Gleason score and a prostate-restricted cancer may enter a phase of “watchful waiting” in which treatment is withheld until signs of progression are identified. However, these prognostic indicators do not accurately predict clinical outcome for individual patients.
Unlike many cancer types, specific patterns of oncogene expression have not been consistently identified in prostate cancer progression, although a number of candidate genes and pathways likely to be important in individual cases have been identified (Tomlins et al., Annu. Rev. Pathol. 1:243-71, 2006). Several groups have attempted to examine prostate cancer progression by comparing gene expression of primary carcinomas to normal prostate. Because of differences in technique, as well as the true biologic heterogeneity seen in prostate cancer, these studies have reported thousands of candidate genes but shared only moderate consensus.
A few genes have emerged including hepsin (HPN; Rhodes et al., Cancer Res. 62:4427-33, 2002), alpha-methylacyl-CoA racemase (AMACR; Rubin et al., JAMA 287:1662-70, 2002) and enhancer of Zeste homolog 2 (EZH2; Varambally et al., Nature 419:624-9, 2002), which have been shown experimentally to have probable roles in prostate carcinogenesis. Most recently, bioinformatic approaches and gene expression methods have been used to identify fusion of androgen-regulated genes including transmembrane protease, serine 2 (TMPRSS2) with members of the erythroblast transformation specific (ETS) DNA transcription factor family (Tomlins et al., Science 310:644-8, 2005). These fusions appear commonly in prostate cancers and have been shown to be prevalent in more aggressive cancers (Attard et al., Oncogene 27:253-63, 2008; Demichelis et al., Oncogene 26:4596-9, 2007; Nam et al., Br. J. Cancer 97:1690-5, 2007). A number of studies have shown distinct classes of cancers separable by their gene expression profiles (Rhodes et al., Cancer Res. 62:4427-33, 2002; Glinsky et al., J. Clin. Invest. 113:913-23, 2004; Lapointe et al., Proc. Natl. Acad. Sci. USA 101:811-6, 2004; Singh et al., Cancer Cell 1:203-9, 2002; Yu et al., J. Clin. Oncol. 22:2790-9, 2004). In addition, a number of gene expression studies have been performed looking for gene dysregulation in metastatic prostate cancers when compared to normal (healthy) prostate tissue (Varambally et al., Nature 419:624-9, 2002; Lapointe et al., Proc. Natl. Acad. Sci. USA 101:811-6, 2004; LaTulippe et al., Cancer Res. 62:4499-506, 2002). One factor impacting clinical utility of gene expression analyses is the fact that most samples available for validation exist only as formalin fixed paraffin embedded (FFPE) tissues. In contrast, many of the cDNA microarray studies have used snap frozen tissues (Bibikova et al., Genomics 89:666-72, 2007; van't Veer et al., Nature 415:530-6, 2002). There are a range of blood proteins whose levels change in subjects as prostate cancer develops. Some of these are produced by prostate cancer cells themselves, while others are produced by cells of non-prostate origin.
PSA is a class of protein that is produced by both healthy and cancerous prostate tissues. Another class of proteins that change as prostate cancer develops are immune system products. These can be divided into immune specific products, such as antibodies, and immune nonspecific products, such as growth factors and acute phase proteins.
Acute-phase proteins are a class of proteins whose plasma concentrations increase in response to inflammation. In response to injury and cancer cell growth, local inflammatory cells (neutrophils, granulocytes and macrophages) secrete a number of cytokines into the bloodstream, most notable of which are the interleukins IL-1, IL-6 and IL-8, and TNF-α. As a result, acute phase proteins such as C-reactive protein (CRP) may be produced in the liver and secreted into the blood. CRPs physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system via the Clq complex. Serum amyloid A proteins are another class of acute phase proteins and are a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma that increase in levels in response to inflammatory stimuli.
Anti-nuclear antibodies (ANA) are autoantibodies directed against the contents of the cell nucleus. They are present in higher than normal numbers in autoimmune disease. The ANA test measures the pattern and amount of autoantibody which can attack the body's tissues as if they were foreign material. Autoantibodies are present in low titers in the general population, but in about 5% of the population their concentration is increased, and about half of this 5% have an autoimmune disease.
Some antigens are found predominantly associated with cancer cells and are termed cancer antigens. CA-125 (cancer antigen 125 or carbohydrate antigen 125; also known as mucin 16 or MUC16) is a member of the mucin family of glycoproteins which may be elevated in the blood of some patients with pancreatic or breast cancer. Carcinoembryonic antigen (CEA) is a type of protein molecule that can be found in many different cells of the body, but is typically associated with certain cancers and the developing fetus. The word “carcinoembryonic” reflects the fact that CEA is produced by some cancers and by the developing fetus.
Typical over-reactions of the host immune response and delayed hypersensitivity reactions are represented by inflammatory infiltrates from T-lymphocytes (CD4+ T helper/inducer cells and CD8+ T cytotoxic/suppressor cells), which are distributed variously between the epithelial and stromal components. Some men with symptoms of chronic prostatitis have evidence of a proliferative CD4/T-cell response to PSA. T-lymphocytes secrete inflammatory mediators such as the complement components C3, C4 and IL-6 in the serum. The concentrations of these markers decrease with the relief of prostatitis symptoms.
The concept of using more than one biomarker to diagnose prostate cancer to improve the accuracy of the PSA test and to increase its prognostic value has been analyzed by a number of investigators. Patterns of biomarker levels in serum have been compared to cancer staging tools as the T (type) classification, N (node) classification, Gleason score and prostate specific antigen (PSA) value before therapy and disease grade. Serum levels of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), chromogranin A (CHGA), serum calcium, and hemoglobin platelet count indicators that add to the accuracy of a PSA value are used by physicians to assess treatment modalities but have not been widely used for prognostic assessment. Large retrospective clinical studies have been undertaken to determine whether disease-specific survival assessed using univariate and multivariate Cox's proportional hazards model analyzes is correlated with patterns of biomarker expression before, during and after treatment.
New prognostic indices clinically applicable for patients with metastatic or late stage (Stage IV) prostate cancer are needed to assess efficacy of treatments because prostate-specific antigen (PSA) tests fail to reflect the prognostic outcome. The PSA/prostatic acid phosphatase (PAP) ratio has been tested as a prognostic index while serum levels of CRP, PSA, CHGA and ALP have been shown to increase and haemoglobin levels have been shown to decrease with advancing extent of disease (EOD) on bone scan EOD grade, and have variously been associated with disease-specific survival. However none of these biomarkers have entered routine diagnostic or prognostic use for prostate cancer.
There thus remains a need in the art for an accurate test for all forms of prostate cancer.
The present invention provides a minimally invasive test that can be employed to detect prostate cancers, to distinguish prostate cancers from benign prostate hypertrophy (BPH) and prostatitis, and to detect prostate cancers in asymptomatic men whose prostate cancer may produce low levels of PSA, with high sensitivity and specificity. The disclosed methods detect multiple biomarkers and correlate their expression levels with the presence or absence of different prostatic diseases and/or stages of prostate cancer. In certain embodiments, the methods utilize patterns of expression of biomarkers to distinguish between indolent cancers which have a low likelihood of progressing to a lethal disease, and more aggressive forms of prostate cancer which are life threatening and require treatment.
In one aspect, the present disclosure provides methods for diagnosing the presence of prostate cancer in a subject, comprising: (a) detecting the levels of expression of the polypeptide biomarkers prostate specific antigen (PSA), kallikrein-2 (KLK2), C reactive protein (CRP) and chromogranin A (CHGA) in a biological sample obtained from the subject; and (b) comparing the levels of expression of the polypeptide biomarkers in the biological sample with predetermined threshold values, wherein levels of expression of at least two, at least three or at least four of the plurality of polypeptide biomarkers above the predetermined threshold values are indicative of the presence of prostate cancer in the subject. In certain embodiments, the disclosed methods further comprise detecting the level of expression of at least one additional polypeptide biomarker selected from the group consisting of: prostatic acid phosphatase (PAP), lactate hydrogenase (LDH) and bone alkaline phosphatase.
The polypeptide sequences for PSA, KLK-2, CRP, CHGA, PAP, LDH and bone alkaline phosphatase are provided in SEQ ID NO: 1-13. In certain embodiments, the methods disclosed herein include detecting the level of expression of a polypeptide comprising a sequence of SEQ ID NO: 1-13, or a variant thereof, as defined herein.
In a related aspect, methods for diagnosing the presence of different types and/or stages of prostate cancer are provided. In one embodiment, methods for diagnosis of early stage prostate cancer in a subject are provided, such methods comprising detecting the expression levels of at least PSA and CRP in a biological sample obtained from the subject, wherein levels of expression of PSA and CRP above predetermined threshold values are indicative of the presence of early stage prostate cancer in the subject. In another embodiment, methods for diagnosing the presence of asymptomatic prostate cancer in a subject are provided, such methods comprising detecting the levels of expression of at least PSA and KLK2 in a sample obtained from the subject, wherein levels of expression of PSA and KLK2 above predetermined threshold values indicate the presence of asymptomatic prostate cancer in the subject. In yet a further embodiment, methods for diagnosing the presence of metastatic prostate cancer in a patient are provided, such methods comprising detecting the levels of expression of PSA, CRP, CHGA and bone alkaline phosphatase in a biological sample obtained the subject, wherein levels of expression of PSA, CRP, CHGA and bone alkaline phosphatase above the predetermined threshold values indicate the presence of metastatic prostate cancer in the subject.
Biological samples that can be effectively employed in the disclosed methods include, but are not limited to, urine, blood, serum, and biopsy tissue.
In certain embodiments, the expression levels of the polypeptide biomarkers employed herein are quantified by immunoassay, such as enzyme-linked immunoassay (ELISA) technology. In specific embodiments, the levels of expression of the polypeptide biomarkers are determined by contacting the biological sample with antibodies, or antigen binding fragments thereof, that selectively bind to the polypeptide biomarkers; and detecting binding of the antibodies, or antigen binding fragments thereof, to the polypeptide biomarkers. The binding agents employed in the disclosed methods and compositions are preferably labeled with a detectable moiety.
For example, the level of a polypeptide biomarker in a sample can be assayed by contacting the biological sample with an antibody, or antigen binding fragment thereof, that selectively binds to the target biomarker (referred to as a capture molecule or antibody), and detecting the binding of the antibody, or antigen-binding fragment thereof, to the polypeptide biomarker. The detection is generally performed using a second antibody to bind to the capture antibody complexed with its target biomarker. A target biomarker can be an entire protein, or a variant or modified form thereof. ELISA kits for the detection of biomarkers as described herein are commercially available and include pre-coated strip plates, biotinylated secondary antibody, standards, controls (where applicable), buffers, streptavidin-horse radish peroxidise (HRP), tetramethyl benzidine (TMB), stop reagents, and detailed instructions for carrying out the tests including performing standards.
The present disclosure also provides methods for the detection of prostate cancer in a subject wherein the levels of expression of the polypeptide biomarkers in a biological sample are determined simultaneously. For example, in one embodiment, methods for diagnosing the presence of prostate cancer in a subject are provided that comprise: (a) contacting a biological sample obtained from the subject with a plurality of binding agents that selectively bind to a plurality of polypeptide biomarkers disclosed herein for a period of time sufficient to form binding agent-polypeptide biomarker complexes; (b) detecting binding of the binding agents to the plurality of polypeptide biomarkers, thereby determining the levels of expression of the polypeptide biomarkers in the biological sample; and (c) comparing the levels of expression of the plurality of polypeptide biomarkers in the biological sample with predetermined threshold values, wherein levels of expression of at least three of the plurality of polypeptide biomarkers above the predetermined threshold values indicates the presence of prostate cancer in the subject. Examples of binding agents that can be effectively employed in such methods include, but are not limited to, antibodies or antigen-binding fragments thereof.
In a further aspect, the present disclosure provides compositions that can be employed in the disclosed methods. In certain embodiments, such compositions a solid substrate and a plurality of binding agents immobilized on the substrate, wherein each of the binding agents is immobilized at a different, indexable, location on the substrate and the binding agents selectively bind to a plurality of polypeptide biomarkers disclosed herein. In embodiment, the binding agents selectively bind to a plurality of polypeptide biomarkers comprising PSA, KLK2, CRP and CHGA. In other embodiments, such compositions additionally comprise binding agents that selectively bind to at least one polypeptide biomarker selected from PAP, LDH and bone alkaline phosphatase. Binding agents that can be employed in such compositions include, but are not limited to, antibodies, or antigen-binding fragments thereof.
In a related aspect, methods for diagnosing the presence of prostate cancer in a subject are provided, such methods comprising: (a) contacting a biological sample obtained from the subject with a composition disclosed herein for a period of time sufficient to form binding agent-polypeptide biomarker complexes; (b) detecting binding of the plurality of binding agents to the plurality polypeptide biomarkers, thereby determining the levels of expression of the plurality of polypeptide biomarkers in the biological sample; and (c) comparing the levels of expression of the plurality of polypeptide biomarkers in the biological sample with predetermined threshold values, wherein levels of expression of at least two of the plurality of polypeptide biomarkers above the predetermined threshold values indicates the presence of prostate cancer in the subject.
In yet another aspect, the present disclosure provides compositions comprising a solid substrate and a plurality of polypeptide biomarkers disclosed herein immobilized on the substrate, wherein each of the polypeptide biomarkers is immobilized at a different, indexable, location on the substrate. In certain embodiments, the plurality of polypeptide biomarkers includes PSA, KLK2, CRP and CHGA. In other embodiments, the plurality of polypeptide biomarkers further includes at least one polypeptide biomarker selected from the group consisting of: PAP, LDH and bone alkaline phosphatase.
In a related aspect, methods for diagnosing the presence of prostate cancer in a subject, comprising: (a) contacting a biological sample obtained from the subject with a composition including a plurality of polypeptide biomarkers disclosed herein for a period of time sufficient to form autoantibody-polypeptide biomarker complexes; (b) detecting the autoantibody-polypeptide biomarker complexes, thereby determining the levels of autoantibodies in the biological sample; and (c) comparing the levels of autoantibodies in the biological sample with predetermined threshold values, wherein levels of autoantibodies against at least two of the plurality of polypeptide biomarkers above the predetermined threshold values indicates the presence of prostate cancer in the subject.
In another aspect, kits for diagnosing the presence or absence of prostate cancer in a subject are provided, such kits comprising binding agents that specifically bind to the polypeptide biomarkers disclosed herein and instructions for their use.
As used herein, the term “biomarker” refers to a molecule that is associated either quantitatively or qualitatively with a biological change. Examples of biomarkers include polypeptides, proteins or fragments of a polypeptide or protein; and polynucleotides, such as a gene product, RNA or RNA fragment; and other body metabolites.
As used herein, the term “antigen” refers to a synthetic peptide, polypeptide, protein or fragment of a polypeptide or protein, or other molecule which elicits an antibody response in a subject, or is recognized and bound by an antibody.
As used herein, the terms “specific binding” or “specifically binding” when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the epitope) on the protein.
As used herein, the terms “binding agent specific for” or “binding agent that specifically binds” refers to an agent that binds to a polypeptide biomarker and does not significantly bind to unrelated proteins. Examples of binding agents that can be effectively employed in the disclosed methods include, but are not limited to, proteins and antibodies, such as monoclonal or polyclonal antibodies, or antigen-binding fragments thereof. In certain embodiments, the binding agent binds the polypeptide biomarker with an affinity constant of, for example, greater than or equal to about 1×10−6 M.
As used herein, the term “subject” refers to a mammal, preferably a human, who may or may not have prostate cancer. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
As used herein, the term “healthy male” refers to a male who has a PSA level in serum of less than 1.0ng/ml, no evidence of prostate gland abnormality following a DRE and no clinical symptoms of prostatic disorders. Subjects who have no evidence of disease outside the prostate gland but whose PSA is climbing are termed “rising PSA” and subjects who are receiving no further treatments are described as being on “watchful waiting”.
As used herein, the term “asymptomatic male” refers to a male who has a PSA level in serum of greater than 8ng/ml, which is considered indicative of prostate cancer, but whose DRE is inconclusive and who has no symptoms of clinical disease.
The term “benign prostate hypertrophy” (BPH) refers to a prostatic disease with a non-malignant growth of epithelial cells in the prostate gland and the term “prostatitis” refers to another prostatic disease of the prostate, usually due to a microbial infection of the prostate gland. Both BPH and prostatitis can result in increased PSA levels.
As used herein, the term “metastatic prostate cancer” refers to prostate cancer which has spread beyond the prostate gland to a distant site, such as lymph nodes or bone.
As used herein, the term “biopsy tissue” refers to a sample of tissue (e.g., prostate tissue) that is removed from a subject for the purpose of determining if the sample contains cancerous tissue. The biopsy tissue is then examined (e.g., by microscopy) for the presence or absence of cancer.
As used herein, the term “sample” is used herein in its broadest sense to include a sample, specimen or culture obtained from any source. Biological samples include blood products (such as plasma, serum, whole blood and peripheral blood mononuclear cells
(PBMCs)), urine, saliva and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formalin, snap frozen, cytological processing, etc.).
As used herein, the term “predetermined threshold value of expression” of a biomarker refers to the level of expression of the same biomarker (generally expressed in ng/ml) in a corresponding control/normal sample or group of control/normal samples obtained from normal, or healthy, males, i.e. males who do not have prostate cancer.
As used herein, the term “altered level of expression” of a biomarker in a test biological sample refers to a level that is either below or above the predetermined threshold value of expression for the same biomarker and thus encompasses either high (increased) or low (decreased) expression levels.
As used herein, the term “oligonucleotide specific for a biomarker” refers to an oligonucleotide that specifically hybridizes to a polynucleotide biomarker or a polynucleotide encoding a polypeptide biomarker disclosed herein, and that does not significantly hybridize to unrelated polynucleotides. In certain embodiments, the oligonucleotide hybridizes to the polynucleotide of interest under stringent conditions, such as, but not limited to, prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
As used, herein the term “polynucleotide(s),” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
The term “prognosis” or “providing a prognosis” refers to providing information regarding the likely impact of the presence of prostate cancer (e.g., as determined by the diagnostic methods) on a subject's future health (e.g., the risk of metastasis).
As outlined above, the present disclosure provides methods for detecting the presence or absence of prostate cancer in a subject, determining the stage of the prostate cancer and/or the phenotype of the prostate cancer, and/or monitoring treatment of prostate cancer by determining the levels of specific combinations of biomarkers in a biological sample obtained from the subject.
The disclosed methods employ at least four biomarkers selected from those shown in Table 1 below. In one embodiment, the disclosed methods comprise determining the expression levels of PSA, KLK2, CRP and CHGA in a biological sample taken from a subject, and comparing the expression levels with a predetermined threshold value. In other embodiments, the methods comprise determining the expression levels of PSA, KLK2, CRP and CHGA, and at least one additional biomarker selected from PAP, bone alkaline phosphatase and LDH. In certain embodiments, the methods comprise determining the expressions levels of each of the seven biomarkers shown in Table 1. These biomarkers were initially identified through analysis of publicly available prostate cancer microarray databases.
The disclosed methods can be employed to diagnose the presence of prostate cancer in subjects with early stage prostate cancer; subjects who have had surgery to remove the prostate (radical prostatectomy); subjects who have had radiation treatment for prostate cancer; subjects who are undergoing, or have completed, androgen ablation therapy; subjects who have become resistant to hormone ablation therapy; and/or subjects who are undergoing, or have had, chemotherapy.
In certain embodiments, the biomarkers disclosed herein are expressed in subjects with prostate cancer at levels that are at least two-fold higher or lower than, or at least two standard deviations above or below, the mean level in normal, healthy individuals, or are at least two-fold higher or lower than, or at least two standard deviations above or below, a predetermined threshold of expression.
In certain embodiments, panels of isolated prostate cancer polypeptide biomarkers are provided that include a plurality of polypeptide sequences (for example at least two, three, four, five, six or seven sequences) selected from SEQ ID NO: 1-13. In related embodiments, panels of binding agents, such as antibodies or antibody fragments, that are specific for the disclosed polypeptide biomarkers are provided, together with panels of oligonucleotides that specifically hybridize to nucleic acid molecules that encode the disclosed polypeptide biomarkers, or that specifically hybridize to the disclosed polynucleotide biomarkers.
All of the biomarkers and binding agents disclosed herein are isolated and purified, as those terms are commonly used in the art. Preferably, the biomarkers and binding agents are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.
In certain embodiments, the binding agents and/or oligonucleotides employed in the disclosed methods specifically bind to a variant of a polypeptide biomarker or polynucleotide biomarker disclosed herein. As used herein, the term “variant” comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a sequence disclosed herein. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
In addition to exhibiting the recited level of sequence identity, variants of the disclosed polypeptide biomarkers are preferably themselves expressed in subjects with prostate cancer at levels that are higher or lower than the levels of expression in normal, healthy individuals.
Variant sequences generally differ from the specifically identified sequence only by conservative substitutions, deletions or modifications. As used herein, a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
Polypeptide and polynucleotide sequences may be aligned, and percentages of identical amino acids or nucleotides in a specified region may be determined against another polypeptide or polynucleotide sequence, using computer algorithms that are publicly available. The percentage identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity.
Two exemplary algorithms for aligning and identifying the identity of polynucleotide sequences are the BLASTN and FASTA algorithms. The alignment and identity of polypeptide sequences may be examined using the BLASTP algorithm. BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences. The FASTA and FASTX algorithms are described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson, Methods in Enzymol. 183:63-98, 1990. The FASTA software package is available from the University of
Virginia, Charlottesville, Va, 22906-9025. The FASTA algorithm, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants. The readme files for FASTA and FASTX
Version 2.0x that are distributed with the algorithms describe the use of the algorithms and describe the default parameters.
The BLASTN software is available on the NCBI anonymous FTP server and is available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894. The BLASTN algorithm
Version 2.0.6 [Sep. 10, 1998] and Version 2.0.11 [Jan. 20, 2000] set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, is described at NCBI's website and in the publication of Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, 1997.
In an alternative embodiment, variant polypeptides are encoded by polynucleotide sequences that hybridize to a disclosed polynucleotide under stringent conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM.
Hybridization temperatures can be as low as 5° C., but are generally greater than about 22° C., more preferably greater than about 30° C., and most preferably greater than about 37° C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. An example of “stringent conditions” is prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
An “antigen-binding fragment” of an antibody refers to the part of the antibody that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains.
Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as the technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976), and improvements thereto. These methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity. Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding antibodies employed in the disclosed methods may be isolated and sequenced using conventional procedures. Recombinant antibodies, antibody fragments, and/or fusions thereof, can be expressed in vitro or in prokaryotic cells (e.g. bacteria) or eukaryotic cells (e.g. yeast, insect or mammalian cells) and further purified as necessary using well known methods.
Antibodies may also be derived from a recombinant antibody library that is based on amino acid sequences that have been designed in silico and encoded by polynucleotides that are synthetically generated. Methods for designing and obtaining in si/ico-created sequences are known in the art (Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J. Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).
Digestion of antibodies to produce antigen-binding fragments thereof can be performed using techniques well known in the art. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the “F(ab)” fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the “F(ab′)2” fragment, which comprises both antigen-binding sites. “Fv” fragments can be produced by preferential proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980)).
Antibody fragments that specifically bind to the polypeptide biomarkers disclosed herein can also be isolated from a library of scFvs using known techniques, such as those described in U.S. Pat. No. 5,885,793.
A wide variety of expression systems are available in the art for the production of antibody fragments, including Fab fragments, scFv, VL and VHS. For example, expression systems of both prokaryotic and eukaryotic origin may be used for the large-scale production of antibody fragments. Particularly advantageous are expression systems that permit the secretion of large amounts of antibody fragments into the culture medium. Eukaryotic expression systems for large-scale production of antibody fragments and antibody fusion proteins have been described that are based on mammalian cells, insect cells, plants, transgenic animals, and lower eukaryotes. For example, the cost-effective, large-scale production of antibody fragments can be achieved in yeast fermentation systems. Large-scale fermentation of these organisms is well known in the art and is currently used for bulk production of several recombinant proteins.
Antibodies that bind to the polypeptide biomarkers employed in the present methods, together with ELISA kits that employ such antibodies for the detection of the biomarkers employed herein, are well known to those of skill in the art and are available commercially.
In certain embodiments, the expression level of one or more polypeptide biomarkers disclosed herein is determined using a binding agent, such as a protein, antibody or antibody fragment, that specifically binds to the polypeptide biomarker of interest, for example in an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, antibody array, Western blot, immunohistochemical, immunoprecipitation or immunofluoresence assay. Methods for performing such assays are well known to those of skill in the art.
In one ELISA method, a first, or capture, binding agent, such as an antibody that specifically binds the biomarker of interest, is immobilized on a suitable solid phase substrate or carrier. The test biological sample is then contacted with the capture antibody and incubated for a desired period of time. After washing to remove unbound material, a second, detection, antibody that binds to a different, non-overlapping, epitope on the biomarker is then used to detect binding of the polypeptide biomarker to the capture antibody. The detection antibody is preferably conjugated, either directly or indirectly, to a detectable moiety. Examples of detectable moieties that can be employed in such methods include, but are not limited to, cheminescent and luminescent agents; fluorophores such as fluorescein, rhodamine and eosin; radioisotopes; colorimetric agents; and enzyme-substrate labels, such as biotin.
In another embodiment, the ELISA is a competitive binding assay, wherein labeled biomarker is used in place of the labeled detection antibody, and the labeled biomarker and any unlabeled biomarker present in the test sample compete for binding to the capture antibody. The amount of biomarker bound to the capture antibody can be determined based on the proportion of labeled biomarker detected.
Solid phase substrates, or carriers, that can be effectively employed in such assays are well known to those of skill in the art and include, for example, 96 well microtiter plates, glass, paper, and microporous membranes constructed, for example, of nitrocellulose, nylon, polyvinylidene difluoride, polyester, cellulose acetate, mixed cellulose esters and polycarbonate. Suitable microporous membranes include, for example, those described in US Patent Application Publication no. US 2010/0093557 Al. Methods for the automation of immunoassays are well known in the art and include, for example, those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750 and 5,358,691.
The presence of several different polypeptide biomarkers in a test sample can be detected simultaneously using a multiplex assay, such as a multiplex ELISA. Multiplex assays offer the advantages of high throughput, a small volume of sample being required, and the ability to detect different proteins across a board dynamic range of concentrations. In certain embodiments, such methods employ an array, wherein multiple binding agents (for example capture antibodies) specific for multiple biomarkers are immobilized on a substrate, such as a membrane, with each capture antibody being positioned at a specific, pre-determined, location on the substrate. Methods for performing assays employing such arrays include those described, for example, in US Patent Application Publication nos. US2010/0093557A1 and US2010/0190656A1, the disclosures of which are hereby specifically incorporated by reference.
Multiplex arrays in several different formats based on the utilization of, for example, flow cytometry, chemiluminescence or electron-chemiluminesence technology, are well known in the art. Flow cytometric multiplex arrays, also known as bead-based multiplex arrays, include the Cytometric Bead Array (CBA) system from BD Biosciences (Bedford, Mass.) and multi-analyte profiling (xMAP®) technology from Luminex Corp. (Austin, Tex.), both of which employ bead sets which are distinguishable by flow cytometry. Each bead set is coated with a specific capture antibody. Fluorescence or streptavidin-labeled detection antibodies bind to specific capture antibody-biomarker complexes formed on the bead set. Multiple biomarkers can be recognized and measured by differences in the bead sets, with chromogenic or fluorogenic emissions being detected using flow cytometric analysis.
In an alternative format, a multiplex ELISA from Quansys Biosciences (Logan, UT) coats multiple specific capture antibodies at multiple spots (one antibody at one spot) in the same well on a 96-well microtiter plate. Chemiluminescence technology is then used to detect multiple biomarkers at the corresponding spots on the plate.
The expression level of one or more polypeptide biomarkers in a biological sample can also be determined by mass spectrometry, in particular liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), using methods well known to those of skill in the art.
The expression levels of one or more polynucleotide biomarkers in a biological sample can be determined, for example, using one or more oligonucleotides that are specific for the biomarker. For example, the levels of mRNA corresponding to a prostate cancer biomarker disclosed herein can be detected using oligonucleotides in Southern hybridizations, in situ hybridizations, and quantitative real-time PCR amplification (qRT-PCR). A plurality of oligonucleotides specific for a plurality of biomarkers can be employed in an array format wherein each oligonucleotide is immobilized at a pre-determined location on a substrate, such as nitrocellulose membrane. Methods for performing such assays are well known to those of skill in the art.
The oligonucleotides employed in the disclosed methods are generally single-stranded molecules, such as synthetic antisense molecules or cDNA fragments, and are, for example, 6-60 nt, 15-30 or 20-25 nt in length.
Oligonucleotides specific for a polynucleotide that encodes a polypeptide biomarker disclosed herein are prepared using techniques well known to those of skill in the art. For example, oligonucleotides can be designed using known computer algorithms to identify oligonucleotides of a defined length that are unique to the polynucleotide, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. Oligonucleotides can be synthesized using methods well known to those in the art. For use in array formats, the oligonucleotides may be synthesized directly on the surface of a substrate. Oligonucleotides specific for the prostate cancer biomarkers disclosed herein are known in the art and are commercially available.
In certain embodiments, the oligonucleotides are labeled using one or more detectable moieties. DNA or mRNA isolated from a biological sample is contacted with the labeled oligonucleotides under conditions that allow for formation of hybridization complexes, and the amount of label associated with the hybridization complexes is measured and compared to a standard value.
In one method, the expression level of one or more prostate cancer biomarkers disclosed herein is determined by first collecting urine from a subject following DRE or prostate massage via a bicycle or exocycle. RNA is isolated from the urine sample and amplified using known techniques, such as those described by Laxman et al. (Neoplasia 2006, 8:885-8) and the expression level of mRNA corresponding to the biomarker is determined by, for example, quantitative PCR or RT-PCR using one or more oligonucleotides specific for the biomarker of interest.
For tests involving alterations in RNA expression levels, it is essential to ensure adequate standardization. Accordingly, in tests such as quantitative real time PCR or small scale oligonucleotide microarrays, at least one expression standard selected is employed.
The present disclosure further provides diagnostic panels including a plurality of binding agents (for example, antibodies) or oligonucleotides that are specific for a plurality (for example, two, three, four, five, six or seven) of the prostate cancer biomarkers disclosed herein. In certain embodiments, such panels are in the form of arrays, or microarrays, in which the binding agents or oligonucleotides are immobilized at specific locations on a substrate. The binding agents and oligonucleotides are preferably labeled such that the size and signal intensity of each labeled complex formed between the biomarker and the binding agent/oligonucleotide are individually distinguishable. Alternatively, diagnostic panels are provided in which the biomarkers disclosed herein are immobilized at specific locations on a substrate. The biomarkers are preferably labeled such that the size and signal intensity of labeled complexes formed between the biomarkers and auto-antibodies present in a biological sample are individually distinguishable. Kits comprising such diagnostic panels and instructions for their use are also provided. Such kits can also include other components required to carry out the assay, such as buffers, preservatives, wash solutions, etc.
The following examples are intended to illustrate, but not limit, this disclosure.
Human ethics approval for the studies described herein was obtained from the Northern X Regional Ethics Committee of New Zealand. A total of 102 blood samples were obtained from subjects who were either volunteer donor healthy males or prostate cancer patients. The distribution of donors with respect to prostate disease status and age is shown in Table 2 below.
Blood samples obtained from the subjects described above were screened for levels of protein biomarkers in serum using commercially available ELISA diagnostic kits purchased from Holzel Diagnostika, Germany. Assays were performed in 96 well microtitre plates and each plate included the relevant standards supplied by the manufacturer to determine a standard curve. The linear portion of the standard curve was selected for determining scores using linear-log or log-log plots of the standard curve data. Samples for analyzes were diluted to meet the detection ranges for the ELISAs.
The protein and gene expression biomarkers selected for use in the methods disclosed herein are summarized above in Table 1. Typical standard curves for A) PSA, B) CRP and C) CHGA for quantification of enzyme linked immunoassays are shown in
The results of specific ELISAs performed to measure A) KLK2, B) CRP, C) CHGA, D) PAP, E) LDH and F) bone alkaline phosphatase levels in individual donor serum samples are shown in
As PSA (KLK3) and KLK2 are structurally related and are known to cross react immunologically, these proteins share common epitopes. To ensure that these were not influencing measurements of either PSA or KLK2 in donor serum, 15 donor samples with high PSA values were selected and ELISAs were performed side by side to compare whether KLK2 values increased with the high PSA values, indicating cross reactivity between the capture antibodies for KLK2 and PSA. As shown in
The term extracted nuclear antigens (ENAs) originally referred to proteins found in a saline extract of mammalian cell nuclei. These proteins are ribonucleoproteins and are commonly used to screen for autoimmune disorders, such as mixed connective tissue disease, Sjogren's syndrome and systemic lupus erythematosus. In order to compare autoimmune responses in prostate cancer subjects to inflammatory responses, antibody levels to ENA and C reactive protein levels in the same serum samples were compared.
The data depicted in
Donor serum samples were obtained from subjects who had a prostatectomy more than one year prior to donating the sample (n=21; 55-69 years) and subjects who presented with metastatic prostate cancer (n=42; 62-78 years).
The metastatic prostate cancer subjects fell into two groups, referred to herein as “Metastatic Group A” and “Metastatic Group B”. Group A (n=12; 42-73 years) were males who had undergone hormone deprivation therapy, were considered hormone resistant and were commencing, or had commenced, chemotherapy within one year of providing a sample for this study. Chemotherapy included different regimes for each patient with some having ketoconazole, Leukine® or taxotere treatment (
The levels of PSA, KLK2, CRP, CHGA, PAP, LAH and bone alkaline phosphatase (BAP) in serum from the subjects, as determined by ELISA, are shown in Table 5 below. As can be seen from the data, distinct patterns of increased CRP, CHGA and bone alkaline phosphatase (BAP) levels were observed in serum from patients with metastatic prostate cancer.
Gene expression analyses were performed using PBMCs from a group of 15 prostate cancer patients and five healthy subjects (three males and two females). The healthy males were determined to be free of prostate cancer and showed no evidence of prostate cancer in a 12 month follow up. Blood was collected at different times over a four month period. The collection times of specific donor samples are shown in Tables 6A and 1B, below, together with an internal code, gender and disease status (CaP=prostate cancer; Norm=healthy subject).
PBMCs were purified by centrifugation over a layer of Fico11™ Hypaque and RNA extractions using recovered cells were all performed with TRIZOL™ (Invitrogen, Cat# 15596-026), according to the manufacturer's instructions. cDNA amplifications were performed with SuperScript VILO cDNA Synthesis Kit (Invitrogen, Cat# 11754-250) containing (10×) SuperScript Enzyme Mix and (5×) VILO Reaction Mix. Briefly, 10 μl of the sample (10 ng/μl final concentration) was added to a PCR tube strip. The master mix was prepared according to the manufacturer's protocol and 10 μl of the master mix was added, mixed by vortexing for a few seconds and centrifuged at 1,000 rpm for a few seconds. Amplification involved the following steps: 10 minutes at 25° C.; 60 minutes at 42° C.; 5 minutes at 85° C.; held at 4° C. Storage of the cDNA was at −20° C.
Quantitative Real Time PCR amplification employed InVitrogen Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Cat. no. 11744-500). Briefly, cDNA samples retrieved from −20° C. were tested in triplicate. Samples were briefly centrifuged, diluted 1:5 with RNase free water, mixed and centrifuged. 2 μl of each cDNA was transferred to appropriate wells of a 384-well microtitre plate. For a single reaction, 5 μl of Syber green master mix plus 0.2 μl of 10 μM forward and reverse specific primers plus were mixed with 2.6 μl of RNase free water. Then 8 μl of each master mix was transferred to appropriate assays in microwells and the plates sealed with optical plastic. After mixing by vortexing, the plates were centrifuged briefly, and then analysed via qRT-PCR (7900 HT Fast Real Time PCR System, Applied Biosystems).
Conditions used for qRT-PCR are described in Table 7, below.
mRNA expression of KLK3 was examined in PBMCs from blood collected from prostate cancer (CaP) and healthy (Normal) donors. The results (shown below in Table 8 and in
mRNA expression of KLK2 was examined in PBMCs from blood collected from prostate cancer (CaP) and healthy (Normal) donors. The results (shown below in Table 9, and in
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, for use in practicing the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
SEQ ID NO: 1-13 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol “n,” conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.
This application is a continuation of U.S. patent application Ser. No. 13/291,980, filed Nov. 8, 2011, the disclosure of which is hereby incorporated by reference.
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| Number | Date | Country | |
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| 20130116133 A1 | May 2013 | US |
| Number | Date | Country | |
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| Parent | 13291980 | Nov 2011 | US |
| Child | 13350480 | US |
