Abstract:
The present invention relates to a method of detecting the presence of cancer by measuring the level of enzyme activity and autoantibodies in the blood of an individual. In particular the present invention relates to methods for measurement of activated cAMP-dependent protein kinase A (PKA) activity and antibodies to PKA, a kit for activated PKA activity measurement, and the use of the measured levels of these analytes for determining the presence of cancer.

Description:
[0001]    This application claims priority of U.S. provisional patent 61/251,769 filed on Oct. 15, 2009. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention is in the field of diagnostics and relates to methods for determining the presence, activity, and concentrations of certain protein biomarkers and their use in determining the presence of cancer. 
       BACKGROUND ART 
       [0003]    The cAMP-dependent protein kinase (PKA) is activated by the binding of cAMP to the regulatory subunit (R), of the molecule and results in the release of the active catalytic kinase subunit (C). Most of the effects of cAMP in eukaryotic systems are the result of phosphorylation of proteins at serine or threonine residues by PKA. There are several isoforms of both subunits of PKA. PKA is localized subcellularly by binding to multidomain scaffolding proteins known as AKAPs which bind to the R subunits of the holoenzyme [1]. More than 50 AKAPs are known which localize PKA in various cell types. The PKA-specific inhibitor (PKI) acts by binding with high affinity to the substrate binding site of the free active catalytic subunit [2]. 
         [0004]    Work done at the National Cancer Institute (NCI) and by others showed that the activity level of PKA is elevated in the plasma of cancer patients, and that anti-PKA antibody levels are elevated in the serum of cancer patients [3-6]. Humphries et al demonstrated that oxidation of PKA inhibited its activity [7]. This inhibition could be overcome by the addition of the reducing agent dithiothreitol. Unexpectedly, the current invention shows that activated (reduced) PKA activity is low in plasma or serum of cancer patients relative to that of controls. 
         [0005]    PSA screening test for prostate cancer 
         [0006]    The PSA (prostate-specific antigen) blood test for prostate cancer is of questionable value as a screening test. In fact the American Cancer Society no longer recommends that men routinely have PSA tests as part of their routine physical examinations [8]. There are several reasons for this lack of support for testing. When prostate cancer is present, the PSA test fails to detect 3 out of 4 cases [9]. In addition, when the PSA test comes back positive, 3 out 4 times it is a false positive—the patient does not have cancer [10] . Nonetheless, patients who have a positive PSA test typically will have a group of 12 or more biopsy samples taken from their prostate to verify if cancer is present. At an average cost of $1,500 per biopsy, the national cost for the 700,000 unnecessary prostate biopsies done each year exceeds $1 billion [11]. Making matters worse, there is a 25% chance that a prostate biopsy will not detect cancer even when cancer is present [12]. Improved patient outcome could be accomplished by replacing a biopsy with a cancer confirmatory blood test, for a savings in health care costs of nearly $900 million annually. 
         [0007]    Mammogram screening test for breast cancer 
         [0008]    Mammograms have a poorer record than PSA tests. While mammograms are purported to detect 85-90% of breast cancers when they are present, the detected tumors on average are 1½ inches in diameter when diagnosed. As for all cancers, earlier detection leads to better patient outcomes. What makes breast cancer screening costly is that an estimated 95% of the positive mammograms are false positives—the patient does not have cancer [13]. A positive mammogram frequently leads to a breast biopsy. A typical needle biopsy costs about $1,500; an invasive surgical biopsy (about ⅓ of all breast biopsies) costs about $5,000. This brings the national cost for the estimated 2 million unnecessary breast biopsies to more than $5.1 billion annually. Improved patient outcome could be accomplished by substituting a cancer confirmatory test for a biopsy, for a savings in health care costs of almost $5 billion annually 
         [0009]    Cancer monitoring tests 
         [0010]    Cancer monitoring tests. Beyond screening tests there are additional blood tests that are used to monitor cancer patients once cancer has been diagnosed. Many of these tests are not specific for cancer or specific for a particular type of cancer, rendering them useless as cancer screening tests. However, they can be an effective means for monitoring cancer treatment and testing for disease recurrence. These tests include CA-15.3 and CA27.29 for breast cancer, CA 125 for ovarian cancer, CEA for colon cancer and PSA for prostate cancer [14]. Other blood tests have been used to determine if a primary cancer has spread to other organs. These tests include assays for metastases to bone (osteoprotegrin), and liver (E-selectin). 
         [0011]    Work done at the National Cancer Institute (NCI) and by others taught that the activity level of PKA is elevated in the plasma of cancer patients, and that anti-PKA antibody levels are elevated in the serum of cancer patients [3-4]. 
       SUMMARY OF INVENTION 
       [0012]    A method is described for determining the presence of cancer in a patient consisting of measuring the activity of activated PKA in a patient sample serum or plasma and determining that the activity of activated PKA is lower than that of a control sample or control population 
         [0013]    In addition a method is provided wherein, in addition to measuring the level of activated PKA in the sample, the level of anti-PKA antibody also is measured. Elevated levels of anti-PKA antibody relative to a control sample or control population plus low levels of activated PKA activity are used to determine the presence of cancer 
         [0014]    Also described is a method for determination of the levels of anti-PKA antibody in human serum or plasma wherein the difference in signal between PKA-coated and uncoated wells is used to correct for non-specific signal. 
         [0015]    Lastly a kit is described for determining the amount of activated PKA activity in a sample. 
       Technical Problem 
       [0016]    There is an important unmet need for non-invasive cancer screening tests and tests to verify the results of positive cancer screening tests. Additionally, better non-invasive tests are needed for determining the stage of cancer when diagnosed and for monitoring patient treatment and recovery. An estimated 1.4 million cases of cancer will be diagnosed in the U.S. this year[15]. As the population is aging the number of cancer cases is expected to increase by 19% [16-17]. Approximately 10.8 million people alive today have, or have had, diagnosed cancer [18]. Over 20 million individuals will be screened for breast or prostate cancer this year. 
         [0017]    A blood test that confirmed, monitored or screened for all types of cancer would be of benefit. The test would serve as a universal confirmation test for presumptive positive PSA, Pap smear, and mammogram tests. Additionally it would be important if the test could determine the stage of diagnosed cancer and whether metastasis has occurred. Such a test could be used to monitor patient treatment, test for cancer recurrence, and it could be used by pharmaceutical companies to monitor the efficacy of cancer drugs under development. 
       Solution to the Problem 
       [0018]    In contrast to what would be expected from the work of other investigators [3-4], we have shown that low levels of activated PKA activity are present in serum or plasma of cancer patients relative to the levels in control samples. This decrease in activity can be used to determine the presence of cancer in patients with breast, colorectal, lung, and prostate cancer. In these experiments oxidized PKA in the sample is activated by the inclusion of β-mercaptoethanol or a similar antioxidant in the reaction mixture. Results from such experiments are detailed below. 
       Advantageous Effects of Invention 
       [0019]    Extremely low levels of PKA activity are detectable in non-reduced blood samples making accurate measurement of enzyme activity very difficult. The addition of an antioxidant to the PKA assay activates the enzyme making it much easier to measure the PKA activity and to measure differences, especially decreases, in enzyme activity. Assaying activated PKA activity in blood samples provides identification of individuals who have cancer by virtue of their low levels of activated PKA activity. The detection of elevated levels of anti-PKA antibodies in addition to measurement of low activated PKA activity provides additional evidence that individuals have cancer. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0020]      FIG. 1  is a bar graph of the levels of activity of activated PKA in prostate cancer patients relative to those of age and sex-matched normal controls. 
           [0021]      FIG. 2  is a bar graph of the levels of activity of activated PKA in prostate cancer patients relative to those of age and sex-matched normal controls. 
           [0022]      FIG. 3  is a bar graph of the levels of anti-PKA antibody relative to the levels of activated PKA activity. 
           [0023]      FIG. 4  is a receiver-operator curve (ROC) plot of the levels of activated PKA activity in cancer patients and age and sex-matched controls. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
     Embodiment 1 
       [0024]    Serum samples from patients with early and late-stage cancer were obtained from ProMedDx, LLC. In one embodiment blood samples from prostate cancer patients and normal controls presumably without cancer were assayed for activated PKA activity. In this assay activated PKA in samples was mixed with a defined peptide used as a substrate. Phosphorylation of the peptide was detected using biotinylated phosphoserine antibody, which was in turn was detected in an ELISA format using peroxidase-conjugated to streptavidin. Detection of the bound peroxidase was established using a color-producing peroxidase substrate included in the assay kit. Bovine PKA catalytic unit was used at varying concentrations to develop a standard activity curve. The detail of the assay protocol is described below. 
         [0025]    Modified MESACUP Protein Kinase A Activity Assay
       1. Reference: Kit Instructions   2. Materials
           a. MESACUP Protein Kinase Assay Kit (MBL Code No. 5230)   b. ATP: 10 mM in water
               i. Dissolve 60 mg ATP (Sigma Prod. No. A2383) in 1.0 ml water   ii. Determine the absorbance of a 1/1000 dilution in PBS at 259 nm   iii. Store at −20° C.   iv. Immediately before use dilute to 10 mM based on the absorbance and the molar extinction coefficient (E 259 , pH 7=15,400)   
               c. PKI inhibitor: 0.5 mM in water (Santa Cruz Prod. No. sc-201160)
               i. Dissolve 1 mg in 1.0 ml water   ii. Store at −20° C.   
               d. PKA diluent: 25 mM KH 2 PO 4 , 5 mM EDTA, 150 mM NaCl, 50% (w/v) glycerol, 1 mg/ml BSA, 5 mM (3-mercaptoethanol, pH 6.5   e. PKA catalytic subunit standard:
               i. Dissolve bovine PKA (Sigma Prod. No. P2645) in cold PKA diluent to a final concentration of 1          g/ml   ii. Store at −20° C.   
               f. Peroxidase substrate solution (Sigma Prod. No. T8665)   
           3. Procedure
           a. Prepare samples
               i. Thaw serum samples   ii. Centrifuge 10 minutes at 16,000×g   iii. Collect the clear supernatant   iv. Mix 0.0108 ml supernatant with 0.0012 ml diluent in a dilution plate, two wells per sample   v. Incubate one hour at room temperature   
               b. Prepare calibration curve
               i. Prepare serial ½ dilutions of PKA 20-0.4 ng/ml in PKA diluent   ii. Dispense 0.012 ml per well of a dilution plate   
               c. Prepare reaction buffer to final concentrations of:
               i. 25 mM tris-HCl, pH 7.0   ii. 3 mM MgCl 2      iii. 1 mM ATP   iv. 0 or 0.5 uM PKI   
               d. Add 0.108 ml reaction buffer (with or without PKI) to each sample or calibrator well of the dilution plate   e. Pre-incubate five minutes at 25° C.   f. Transfer 0.100 ml per well to assay plate   g. Incubate 20 minutes at 25° C. with shaking at 750 rpm   h. Add 0.100 ml kit stop solution per well   i. Wash three times with kit wash buffer   j. Add 0.100 ml kit biotinylated anti-phosphoserine per well   k. Incubate 60 minutes at 25° C. with shaking at 750 rpm   l. Wash three times with kit wash buffer   m. Add 0.100 ml kit peroxidase-conjugated streptavidin per well   n. Incubate 60 minutes at 25° C. with shaking at 750 rpm   o. Wash three times with kit wash buffer   p. Add 0.100 ml peroxidase substrate solution per well   q. Incubate 60 minutes at 25° C. with shaking at 750 rpm   r. Add 0.100 ml kit stop solution per well   s. Shake briefly until well mixed   t. Read absorbance at 450 nm   
           4. Calculation of results
           a. Plot absorbance versus concentration of the calibration curve   b. Perform a least squares linear regression on the data to determine the slope and intercept   c. Calculate kinase concentration in the samples
               i. Kinase (ng/ml)=(sample absorbance—intercept)/slope   
               d. Calculate net PKA in the samples
               i. Net PKA (ng/ml)=Kinase (0          M PKI)—Kinase (0.5          M PKI)   
               
               
 
         [0081]    The activity levels of activated PKA in blood from the breast cancer patients were lower (below 4 ng/ml) compared to those for samples from age and sex-matched controls ( FIG. 1 ). 
       Embodiment 2  
       [0082]    Blood samples from breast cancer patients and from age and sex-matched controls presumably without cancer were analyzed for activated PKA activity using the same protocol as used in Embodiment 1. The activity levels of activated PKA in blood from prostate cancer patients were lower (below 4 ng/ml) than those for samples from normal controls ( FIG. 2 ). 
       Embodiment 3 
       [0083]    The same prostate cancer patient samples and related control samples used in Embodiment 1 were tested for anti-PKA antibodies as described below. 
         [0084]    PKA Autoantibody ELISA
       1. Materials
           a. MaxiSorp 96 well polystyrene plates (NUNC Prod. No. 439454)   b. PKA diluent: 25 mM KH 2 PO 4 , 5 mM EDTA, 150 mM NaCl, 50% (w/v) glycerol, 1 mg/ml BSA, 5 mM          -mercaptoethanol, pH 6.5   c. Protein Kinase A (Sigma Prod. No. P2645)
               i. Dissolve in PKA diluent and dilute to 50          g/ml   ii. Store at −20° C.   
               d. Coating buffer: 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4   e. Coating wash buffer: 20 mM HEPES, 150 mM sodium chloride, 30 mM sucrose, pH 7.0 with 0.1% BSA   f. Blocker Casein (Pierce Prod. No. 37532)   g. Assay buffer: 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 with 0.25% BSA and 0.1% Tween 20   h. Assay wash buffer: 10 mM citrate, 150 mM sodium chloride, pH 5.1 with 0.1% Tween 20   i. Detection antibody: peroxidase-conjugated donkey anti-human IgG (H+L) (Jackson Prod. No. 709-035-149), 0.8 mg/ml in 10 mM sodium phosphate, 250 mM sodium chloride, pH 7.6 with 15 mg/ml bovine serum albumin   j. Stop solution: 0.2 M H 2 SO 4  in water   k. HRP substrate solution (Sigma Prod. No. T8665)   
           2. Procedure
           a. Prepare the assay plates
               i. Dilute PKA to 0.5          g/ml in coating buffer (+PKA wells)   ii. Dilute PKA diluent 1/100 in coating buffer (−PKA wells)   iii. Add 0.100 ml either+PKA or−PKA solution per well   iv. Seal and incubate overnight at 4° C.   v. Aspirate to remove the coating solutions   vi. Add 0.200 ml blocking solution per well   vii. Incubate one hour at room temperature   viii. Wash three times with coating wash buffer   ix. Use immediately   
               b. Prepare samples
               i. Thaw serum samples   ii. Centrifuge 10 minutes at 16,000×g   iii. Collect the clear supernatant   iv. Dilute 1/500 in assay buffer   
               c. Add 0.100 ml diluted sample each to the+PKA and−PKA wells   d. Incubate two hours at 25° C. with shaking at 750 rpm   e. Wash three times with assay wash buffer   f. Dilute the detection antibody 1/20,000 in assay buffer   g. Add 0.100 ml per well   h. Incubate one hour at 25° C. with shaking at 750 rpm   i. Wash three times with assay wash buffer   j. Add 0.100 ml HRP substrate solution per well   k. Incubate 20 minutes at 25° C. with shaking at 750 rpm   l. Add 0.100 ml stop solution per well   m. Shake briefly until well mixed   n. Read absorbance at 450 nm   
           3. Calculation of results
           a. Calculate net anti-PKA signal in the samples
               i. Net anti-PKA=Absorbance (+PKA)—Absorbance (−PKA)   
               
               
 
         [0130]    The levels of anti-PKA antibodies in the prostate cancer patient serum relative to the levels of activated PKA activity (anti-PKA antibody/activated PKA activity) were higher than those for samples from age and sex-matched controls ( FIG. 3 ). The activated PKA activities for these samples also were low relative to those of matched controls ( FIG. 1 ). These observations using the same samples used in Embodiment 1 indicate that the results of both assays together can be used to detect the presence of cancer. 
         [0131]    Embodiment 4 
         [0132]    In this embodiment blood samples from 24 patients with prostate, breast, colon, or lung cancer and 24 age- and sex-matched normal controls presumably without cancer were assayed for activated PKA activity. 20 of 24 cancer patients had low activated PKA activity, while 20 of 24 normal controls had high activity. The calculated sensitivity of the assay was 0.83 and the assay specificity was 0.83. Receiver-operator curve analysis of the results indicated that there was a 0.833 correlation of low activated PKA activity with the presence of cancer at a cutoff value of 3.8 ng/ml of activated PKA activity. ( FIG. 4 ). 
       INDUSTRIAL APPLICABILITY 
       [0133]    The measurement of the levels of activated PKA activity can be used to determine the presence of cancer in individuals. In addition the measurement of levels of activated PKA activity and anti-PKA antibody can be used together to indicate the presence of cancer in individuals. 
       REFERENCE SIGNS LIST 
     Citation List 
       [0134]    1. Pearce, L. R., D. Komander, and D. R. Alessi,  The nuts and bolts of AGC protein kinases.  Nat Rev Mol Cell Biol, 2010. 11(1): p. 9-22. 
         [0135]    2. Walsh, D. A., et al.,  The inhibitor protein of the cAMP - dependent protein kinase,  in  Peptides and Protein Phosphorylation,  B. E. Kemp, Editor. 1990, CRC Press, Inc.: Boca Raton, FL. p. 43-84. 
         [0136]    3. Cho, Y. S., Y. N. Lee, and Y. S. Cho-Chung,  Biochemical characterization of extracellular cAMP - dependent protein kinase as a tumor marker.  Biochem Biophys Res Commun, 2000. 278(3): p. 679-84. 
         [0137]    4. Cho, Y. S., et al.,  Extracellular protein kinase A as a cancer biomarker: its expression by tumor cells and reversal by a myristate - lacking Calpha and RIIbeta subunit overexpression.  Proc Natl Acad Sci U S A, 2000. 97(2): p. 835-40. 
         [0138]    5. Nesterova, M. V., et al.,  Autoantibody cancer biomarker: extracellular protein kinase A.  Cancer Res, 2006. 66(18): p. 8971-4. 
         [0139]    6. Cho-Chung, Y. S. and C. Chung,  Autoantibody detection for cancer diagnostics  2005, Govt. of the U.S. of America: United States. 
         [0140]    7. Humphries, K. M., C. Juliano, and S. S. Taylor,  Regulation of cAMP - dependent protein kinase activity by glutathionylation.  J Biol Chem, 2002. 277(45): p. 43505-11. 
         [0141]    8. Rollins, G.,  PSA Testing: Yes, No, Maybe.  Clin Lab News, 2009. 35(6): p. 1, 3-5. 
         [0142]    9. Smith, D. S., P. A. Humphrey, and W. J. Catalona,  The early detection of prostate carcinoma with prostate specific antigen: the Washington University experience.  Cancer, 1997. 80(9): p. 1852-6. 
         [0143]    10. Schroder, F. H., et al.,  Screening and prostate - cancer mortality in a randomized European study.  N Engl J Med, 2009. 360(13): p. 1320-8. 
         [0144]    11. Jemal, A., et al.,  Cancer Statistics,  2008. CA Cancer J Clin, 2008. 58(2): p. 71-96. 
         [0145]    12. Ahmed, H. U., et al.,  Is it time to consider a role for MRI before prostate biopsy?  Nat Rev Clin Oncol, 2009. 6(4): p. 197-206. 
         [0146]    13. Elmore, J. G., et al.,  Screening for Breast Cancer.  JAMA, 2005. 293(10): p. 1245-1256. 
         [0147]    14. Harris, L., et al.,  American Society of Clinical Oncology  2007  update of recommendations for the use of tumor markers in breast cancer.  J Clin Oncol, 2007. 25(33): p. 5287-312. 
         [0148]    15. Jemal, A., et al.,  Cancer Statistics,  2009. CA Cancer J Clin, 2009: p. caac.20006. 
         [0149]    16. Boudreau, A. C., et al.,  Signaling pathway adaptations and novel protein kinase A substrates related to behavioral sensitization to cocaine.  J Neurochem, 2009. 110(1): p. 363-77. 
         [0150]    17. Smith, B. D., et al.,  Future of cancer incidence in the United States: burdens upon an aging, changing nation.  J Clin Oncol, 2009. 27(17): p. 2758-65. 
         [0151]    18.  American Cancer Society. Cancer Facts &amp; Figures  2008. 2008, American Cancer Society: Atlanta.