Method for predicting the risk of getting cancer or diagnosing cancer in a female subject

Abstract

Subject matter of the present invention is a method for predicting the risk of getting cancer in a female subject that does not suffer from cancer or alternatively diagnosing cancer in a female subject comprising: determining the level of Pro-Enkephalin or fragments thereof including Leu-Enkephalin and Met-Enkephalin of at least 5 amino acids in a bodily fluid obtained from said female subject; and correlating said level of Pro-Enkephalin or fragments thereof with a risk for getting cancer, wherein a reduced level is predictive for an enhanced risk of getting cancer or alternatively diagnosing cancer wherein an reduced level is correlated with the diagnosis of cancer.

Claims

1. A method for predicting a risk of getting cancer in a female subject that does not suffer from cancer, wherein said cancer is breast cancer or lung cancer, said method comprising: measuring the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in a sample of serum or plasma obtained from said female subject using an immunoassay that has antibodies or fragments of antibodies that bind to Pro-Enkephalin or said fragments thereof; correlating said level of Pro-Enkephalin or fragments thereof with a risk of said subject for getting breast or lung cancer, wherein a reduced level is predictive for an enhanced risk of getting breast or lung cancer; and treating said subject with an anti-breast cancer or an anti-lung cancer treatment; wherein a reduced level of Pro-Enkephalin or fragments thereof is a level below 100 pmol/l, and wherein said Pro-Enkephalin or fragment thereof is SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, or SEQ ID No. 11 and wherein the immunoassay comprises: a) bringing said serum or plasma sample into contact with a solid phase comprising a bound first antibody or first antibody fragment that binds to Pro-Enkephalin or fragments thereof whereby Pro-Enkephalin or said fragments thereof within said sample react with said bound first antibody or antibody fragment to form a complex bound to said solid phase, b) contacting said solid phase with the bound complex with a second antibody or second antibody fragment, wherein said second antibody or second antibody fragment is labelled with a detectable label, and whereby the labelled second antibody or second antibody fragment binds to said complex, and c) measuring the level of Pro-Enkephalin or fragments thereof in said sample by measuring the amount of labelled second antibody or second antibody fragment bound to the complex on said solid phase.

2. The method according to claim 1, wherein said female subject has never had a history of diagnosis of cancer at the time said sample of serum or plasma is taken from said female subject.

3. The method according to claim 1, wherein said female subject has had a history of diagnosis of cancer and has been cured at the time said sample of serum or plasma is taken from said female subject and the risk of reoccurrence of getting breast cancer is determined or alternatively the reoccurrence of breast cancer is diagnosed.

4. The method according to claim 1, wherein at the time said sample of serum or plasma is taken from said female subject, said female subject has been diagnosed as having a cardiovascular disease or diabetes.

5. The method according to claim 1, further comprising determining at least one clinical parameter selected from age, presence of diabetes mellitus, and currently smoking.

6. The method according to claim 1, wherein said method is used to stratify female subjects into risk groups.

7. The method according to claim 1, wherein said cancer is breast cancer.

8. The method according to claim 1, wherein said correlating is performed by: (a) comparing the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in said sample of serum or plasma obtained from said female subject with the median of the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids an ensemble of pre-determined samples in a population of healthy or apparently healthy subjects, (b) comparing the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in said sample of serum or plasma obtained from said female subject with a quantile of the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids an ensemble of pre-determined samples in a population of healthy or apparently healthy subjects, or (c) calculating the risk by Cox Proportional Hazards analysis or by Risk index calculations using the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in said sample of serum or plasma obtained from said female subject.

9. The method according to claim 1, wherein one of either (a) said bound first antibody or first antibody fragment, or (b) said labelled second antibody or second antibody fragment, binds to a peptide consisting of amino acid sequence 133-140 of Pro-Enkephalin, and the other of (a) said bound first antibody or first antibody fragment, and (b) said labelled second antibody or second antibody fragment, binds to a peptide consisting of amino acid sequence 152-159 of Pro-Enkephalin.

10. The method according to claim 1, wherein said method is performed more than once in order to monitor the risk of getting breast cancer in said female subject or in order to monitor the course of treatment for breast cancer.

11. The method according to claim 10, wherein monitoring is performed in order to evaluate the response of said female subject to preventive and/or therapeutic measures taken.

12. The method according to claim 1, further comprising measuring the level of Pro-Neurotensin 1-117 (SEQ ID No. 17) in a sample of serum or plasma obtained from said female subject using an immunoassay that has antibodies or fragments of antibodies that bind to Neurotensin 1-117 (SEQ ID No. 17); and correlating said level of Pro-Enkephalin or fragments thereof and said level of Pro-Neurotensin 1-117 (SEQ ID No. 17) with a risk for getting breast cancer or lung cancer, wherein a reduced level of Pro-Enkephalin is predictive for an enhanced risk of getting breast cancer or lung cancer, or alternatively diagnosing breast cancer or lung cancer wherein said reduced level of Pro-Enkephalin is correlated with the diagnosis of breast cancer or lung cancer and wherein an increased level of Pro-Neurotensin is predictive for an enhanced risk of getting breast cancer or lung cancer or alternatively diagnosing breast cancer or lung cancer wherein said increased level of Pro-Neurotensin is correlated with the diagnosis of breast cancer or lung cancer.

13. The method according to claim 12, wherein an increased level of Pro-Neurotensin or fragments thereof is a level above 78 pmol/1.

14. The method according to claim 12, wherein said measuring comprises measuring the level of MR-Pro-Enkephalin (SEQ ID No. 6) and measuring the level Pro-Neurotensin 1-117 (SEQ ID No. 17).

15. The method according to claim 12, wherein said cancer is breast cancer.

16. The method according to claim 12, wherein the measuring of the level of Pro-Neurotensin or fragment thereof comprises: a) bringing said sample of serum or plasma into contact with a solid phase comprising a bound third antibody or third antibody fragment that binds to Pro-Neurotensin or said fragment thereof whereby Pro-Neurotensin said fragment thereof within said sample reacts with said bound third antibody or third antibody fragment to form a complex bound to said solid phase, b) bringing said solid phase with the bound complex into contact with a fourth antibody or fourth antibody fragment, wherein said fourth antibody or fourth antibody fragment is labelled with a detectable label, and whereby the labelled fourth antibody or fourth antibody fragment binds to said complex, and c) measuring the level of Pro-Neurotensin or fragment thereof in said sample by measuring the amount of labelled fourth antibody or fourth antibody fragment bound to the complex on said solid phase.

17. The method according to claim 16, wherein one of either (a) said bound third antibody or third antibody fragment, or (b) said labelled fourth antibody or fourth antibody fragment, binds to a peptide consisting of amino acid sequence 1-19 of Pro-Neurotensin, and the other of (a) said bound third antibody or third antibody fragment, and (b) said labelled fourth antibody or fourth antibody fragment, binds to a peptide consisting of amino acid sequence 44-62 of Pro-Neurotensin.

18. The method according to claim 12, wherein one of either (a) said bound first antibody or first antibody fragment, or (b) said labelled second antibody or second antibody fragment, binds to a peptide consisting of amino acid sequence 133-140 of Pro-Enkephalin, and the other of (a) said bound first antibody or first antibody fragment, and (b) said labelled second antibody or second antibody fragment, binds to a peptide consisting of amino acid sequence 152-159 of Pro-Enkephalin.

19. The method according to claim 18, wherein the measuring of the level of Pro-Neurotensin or fragment thereof comprises: a) bringing said sample of serum or plasma into contact with a solid phase comprising a bound third antibody or third antibody fragment that binds to Pro-Neurotensin or said fragment thereof whereby Pro-Neurotensin said fragment thereof within said sample reacts with said bound third antibody or third antibody fragment to form a complex bound to said solid phase, b) bringing said solid phase with the bound complex into contact with a fourth antibody or fourth antibody fragment, wherein said fourth antibody or fourth antibody fragment is labelled with a detectable label, and whereby the labelled fourth antibody or fourth antibody fragment binds to said complex, and c) measuring the level of Pro-Neurotensin or fragment thereof in said sample by measuring the amount of labelled fourth antibody or fourth antibody fragment bound to the complex on said solid phase.

20. The method according to claim 19, wherein one of either (a) said bound third antibody or third antibody fragment, or (b) said labelled fourth antibody or fourth antibody fragment, binds to a peptide consisting of amino acid sequence 1-19 of Pro-Neurotensin, and the other of (a) said bound third antibody or third antibody fragment, and (b) said labelled fourth antibody or fourth antibody fragment, binds to a peptide consisting of amino acid sequence 44-62 of Pro-Neurotensin.

Description

EXAMPLES

Example 1

(1) Development of Antibodies

(2) Peptides

(3) Peptides were synthesized (JPT Technologies, Berlin, Germany).

(4) Peptides/Conjugates for Immunization:

(5) Peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal Cystein residue for conjugation of the peptides to bovine serum albumin (BSA). The peptides were covalently linked to BSA by using Sulfo-SMCC (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

(6) TABLE-US-00004 TABLE1 Peptideforimmunization Pro-Enkephalinsequence (C)DAEEDD(SEQIDNO:26) 119-125 (C)EEDDSLANSSDLLK(SEQ 121-134 IDNO:27) (C)LKELLETG(SEQIDNO:28) 133-140 (C)TGDNRERSHHQDGSDNE(SEQ 139-155 IDNO:29) (C)SDNEEEVS(SEQIDNO:30) 152-159

(7) The antibodies were generated according to the following method:

(8) A BALB/c mouse was immunized with 100 g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 l complete Freund's adjuvant) and 50 g at day 21 and 28 (in 100 l incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 g of the conjugate dissolved in 100 l saline, given as one intraperitonal and one intravenous injection.

(9) Spenocytes from the immunized mouse and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37 C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-supplement]. After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

(10) The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined.

(11) (Lane, R. D. A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secreting hybridomas, J. Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al. Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11-15, (1996)).

(12) Monoclonal Antibody Production

(13) Antibodies were produced via standard antibody production methods (Marx et al., Monoclonal Antibody Production (1997), ATLA 25, 121) and purified via Protein A-chromatography. The antibody purities were >95% based on SDS gel electrophoresis analysis.

(14) Labelling and Coating of Antibodies.

(15) All antibodies were labelled with acridinium ester according the following procedure:

(16) Labelled compound (tracer): 100 g (100 l) antibody (1 mg/ml in PBS, pH 7.4, was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

(17) Solid Phase Antibody (Coated Antibody):

(18) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 g antibody/0.3 ml 100 mmol/l NaCl, 50 mmol/l Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.

(19) Antibody Specificity:

(20) The crossreactivities of the different antibodies are listed in table 2.

(21) TABLE-US-00005 TABLE2 Peptidefor Pre-Pro- Antibody immunization Enkephalin-sequence name (C)DAEEDD(SEQID 119-125 NT-MRPENK NO:26) (C)EEDDSLANSSDLLK 121-134 NM-MRPENK (SEQIDNO:27) (C)LKELLETG(SEQID 133-140 MR-MRPENK NO:28) (C)TGDNRERSHHQDGSDNE 139-155 MC-MRPENK (SEQIDNO:29) (C)SDNEEEVS(SEQID 152-159 CT-MRPENK NO:30)

(22) Antibody cross-reactivities were determined as follows:

(23) 1 ug peptide in 300 l PBS, pH 7.4 was pipetted into Polystyrene tubes and incubated for 1 h at room temperature. After incubation the tubes were washed 5 times (each 1 ml) using 5% BSA in PBS, pH 7.4. Each of the labelled antibodies were added (300 l in PBS, pH 7.4, 800.000 RLU/300 l) an incubated for 2 h at room temperature, After washing 5 times (each 1 ml of washing solution (20 mmol/1 PBS, pH 7.4, 0.1% Triton X 100), the remaining luminescence (labelled antibody) was quantified using the AutoLumat LB 953. MRPENK-peptide (SEQ ID NO. 6) was used as reference substance (100%).

(24) TABLE-US-00006 TABLE 3 antibody TGDNRERSH DAEEDD EEDDSLANSSD LKELLETG HQDGSDNE SDNEEEVS MRPENK peptide (SEQ ID LLK (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID NO: 27) NO: 28) NO: 29) NO: 30) NO. 6) NT- 121 10 <1 <1 <1 100 MRPENK NM- <1 98 <1 <1 <1 100 MRPENK MR- <1 <1 105 <1 <1 100 MRPENK MC- <1 <1 <1 115 <1 100 MRPENK CT- <1 <1 <1 <1 95 100 MRPENK

(25) All antibodies bound the MRPENK peptide (SEQ ID NO. 6), comparable to the peptides which were used for immunization. Except for NT-MRPENK-antibody (10% cross reaction with EEDDSLANSSDLLK) (SEQ ID NO. 27) no antibody showed a cross reaction with MR-PENK peptides not used for immunization of the antibody.

(26) Pro-Enkephalin Immunoassay:

(27) 50 l of sample (or calibrator) was pipetted into coated tubes, after adding labeled antibody (200 l), the tubes were incubated for 2 h at 18-25 C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X-100). Tube-bound labelled antibody was measured by using the Luminumeter LB 953 and a fixed concentration of 1000 pmol/l of MRPENK. The signal (RLU at 1000 pmol MRPENK/l) to noise (RLU without MRPENK) ratio of different antibody combinations is given in table 4. All antibodies were able to generate a sandwich complex with any other antibody. Surprisingly, the strongest signal to noise ratio (best sensitivity) was generated by combining the MR-MRPENK- and CT-MRPENK antibody. Subsequently, we used this antibody combination to perform the MRPENK-immunoassay for further investigations. MR-MRPENK antibody was used as coated tube antibody and CT-MRPENK antibody was used as labelled antibody.

(28) TABLE-US-00007 TABLE 4 Solid phase antibody Labelled NT- NM- MR- MC- CT- antibody MRPENK MRPENK MRPENK MRPENK MRPENK NT- / 27 212 232 <1 MRPENK NM- 36 / 451 487 <1 MRPENK MR- 175 306 / 536 1050 MRPENK MC- 329 577 542 / <1 MRPENK CT- <1 615 1117 516 / MRPENK

(29) Calibration:

(30) The assay was calibrated, using dilutions of synthetic MRPENK (SEQ ID NO. 6), diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 M Amastatin, 100 M Leupeptin, pH 8.0. Pro-Enkephalin control plasma is available at ICI-diagnostics, Berlin, Germany.

(31) FIG. 1 shows a typical Pro-Enkephalin dose/signal curve. Standard curve Pro-Enkephalin.

(32) The assay sensitivity was 20 determinations of 0-calibrator (no addition of MRPENK)+2SD) 5.5 pmol/L.

(33) Population Study

(34) Methods

(35) We measured Pro-Enkephalin in fasting plasma from 2559 female participants of the population based Malm Diet and Cancer Study baseline exam in 1991-1994 (age 586 years and 59% females). We used multivariable adjusted (all traditional cardiovascular risk factors, diabetes risk factors and in analyses of cancer also heredity for cancer) Cox proportional hazards models to relate baseline PENK (hazard ratio per each standard deviation increase of log-transformed PENK) to the time to the first event of each of the studied endpoints during a median follow-up time of more than 12 years. Endpoints were retrieved through the Swedish National Hospital Discharge Registry, the Swedish Myocardial Infarction Registry, the Stroke in Malm Registry and the Swedish Cancer Registry. Retrieval of endpoints through these registries has been validated and found to be accurate (see also Belting et al. Cancer Epidemiol Biomarkers Prev; 1-10. 2012 AACR).

(36) Clinical Characteristics of Females in the Study

(37) TABLE-US-00008 TABLE 5 Descriptive Statistics N Mean Std. Deviation Age at MDCS screening 2559 57.554 5.9403 Systolic blood pressure (mmHg) 2559 140.50 19.311 Diastolic blood pressure (mmHg) 2559 85.65 9.117 body-mass-index (weight/kg kg) 2559 25.5196 4.19083 WAIST (cm) 2559 76.99 10.245 Glucose (mmol/l) 2559 5.0418 1.21798 Triglycerides (mmol/l) 2559 1.2245 .58404 High density lipoprotein (mmol/l) 2559 1.5123 .36949 Low density lipoprotein (mmol/l) 2559 4.2016 1.04762 P-INSULIN 2512 7.223 5.4223

(38) FIG. 2: frequency distribution of Pro Enkephalin in the females population:

(39) The mean value was 47.2 pmol/L, standard deviation=1.2 pmol/L. The x axis is the Logarithmus Naturalis (LN) of the PENK concentration. All results were within the measurement of the assay, the lowest PENK concentration was 9 pmol/L. These results indicating the suitability of the used assay (assay sensitivity 5.5 pmol/L).

(40) PENK and Prediction of Breast Cancer

(41) We assessed the relationship between Pro-Enkephalin and breast cancer (Table 6). There was a strong relationship between Pro-Enkephalin and breast cancer in females. In a fully adjusted model each SD increase of Pro-Enkephalin was associated with a 28.6% risk reduction or each SD of decrease of Pro-Enkephalin (revPENK) was associated with a 40% increased risk of future breast cancer (table 5) and the top versus bottom quartile of Pro-Enkephalin identified a more than 3-fold difference in risk of breast cancer (see table 7 and FIG. 3).

(42) TABLE-US-00009 TABLE 6 Variables in the Equation.sup.D 95.0% CI for Exp(B) B SE Wald df Sig. Exp(B) Lower Upper AGE .007 .016 .228 1 .633 1.007 .977 1.039 SEX .sup.0.sup.a BMI_B .026 .025 1.139 1 .286 1.027 .978 1.077 DM_B .242 .407 .352 1 .553 .785 .354 1.744 HDL_B .044 .252 .031 1 .860 1.045 .638 1.714 LDL_B .001 .090 .000 1 .988 .999 .837 1.191 current_smoker .330 .195 2.886 1 .089 1.392 .950 2.037 HER_CANCER_0 .034 .176 .038 1 .846 1.035 .733 1.461 LNINS .288 .197 2.127 1 .145 .750 .509 1.104 ZscoreLNPENK.sub. .337 .082 16.858 1 .000 .714 .608 .839 females_noCa

(43) TABLE-US-00010 TABLE 7 BREAST CANCER HR per P- Quar- Quar- Quar- Quar- P for 1 SD value tile 4 tile 3 tile 2 tile 1 trend Women 1.40 <0.001 1.0 1.50 2.7 3.6 <0.001 (2140/ (13- (ref) (0.81- (1.7- (2.7- 135) 1.6) 2.1) 3.4) 4.9)

(44) Multivariate Cox proportional Hazards models for baseline Pro-Enkephalin versus incidence of breast cancer.

(45) FIG. 3: Kaplan Meier graphs, illustrating the cumulative breast cancer diagnosis in women Quartile (Q) 1 (below 40.4 pmol/l) quartile 2 (40.4-47.1 pmol/l), quartile 3 (47.2-54.1 pmol/l), quartile 4 (above 54.1 pmol/l). Decreased PENK indicates a long term increased risk of breast cancer development. Since any women with cancer history at day of baseline (blood sampling) were excluded, Pro-Enkephalin is highly predictive for future breast cancer development. Over all, women from Q 1 have a 3.6 times higher risk to develop breast cancer than women from Q 4.

(46) Combination Pro Enkephalin and Pro Neurotensin

(47) Since increasing Pro-Neurotensin recently was shown to be highly predictive for breast cancer, we combined both biomarkers for breast cancer prediction.

EXAMPLES

(48) Pro-Neurotensin Assay

(49) Antibodies were generated as described above. The antibody for labelling (LA) was generated against P-NT 1-19 (H-CSDSEEEMKALEADFLTNMH (SEQ ID NO: 24)) and the solid phase antibody (SPA) was generated against peptide P-NT 44-62 (CNLNSPAEETGEVHEEELVA (SEQ ID NO: 25)).

(50) Immunoassay for the Quantification of Human Pro-Neurotensin

(51) The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.

(52) Labelled compound (tracer): 100 g (100 l) LA (1 mg/ml in PBS, pH 7.4, was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled LA was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified LA was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

(53) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with SPA (1.5 g SPA/0.3 ml 100 mmol/1 NaCl, 50 mmol/1 Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.

(54) Calibration:

(55) The assay was calibrated, using dilutions of Pro-Neurotensin containing human serum. A pool of human sera with high Pro-Neurotensin immunoreactivity (InVent Diagostika, Hennigsdorf, Germany) was diluted with horse serum (Biochrom AG, Deutschland) (assay standards).

(56) The standards were calibrated by use of the human Pro-Neurotensin-calibrator (ICI-Diagnostics, Berlin, Germany). Alternatively, the assay may be calibrated by synthetic or recombinant P-NT 1-117 or fragments thereof (see also Ernst et al., 2006).

(57) ProNT Immunoassay:

(58) 50 l of sample (or calibrator) was pipetted into SPA coated tubes, after adding labeled LA (200 ul), the tubes were incubated for 16-22 h at 18-25 C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X-100). Tube-bound LA was measured by using the Luminumeter LB 953. Results were calculated from the calibration curve.

(59) Combined analysis of Pro-Enkephalin and PNT in the female population:

(60) There was no significant correlation between Pro-Enkephalin and Pro-Neurotensin (p=0.56). In a combined model using both biomarkers, we found them both independent in breast cancer prediction.

(61) In a fully adjusted model each SD increase of PNT was associated with a 49.9% risk increase of future breast cancer. Surprisingly, after adding PNT to the equation, PENK (SEQ ID NO. 1) was even stronger than without PNT and showed for each SD increase of Pro-Enkephalin a 30.8% risk reduction or each SD of decrease of Pro-Enkephalin (revPENK) was associated with a 44.5% increased risk of future breast cancer (table 8).

(62) TABLE-US-00011 TABLE 8 combined analysis of PNT and PENK for breast cancer prediction. Variables in the Equation 95.0% CI for Exp(B) B SE Wald df Sig. Exp(B) Lower Upper AGE .003 .019 .020 1 .888 .997 .960 1.036 current_smoker0 .434 .204 4.505 1 .034 1.543 1.034 2.304 BMI_B .001 .027 .001 1 .979 1.001 .948 1.056 GFR_CG_BSAcorr .005 .008 .357 1 .550 .995 .979 1.011 hrt_curr .730 .201 13.146 1 .000 2.075 1.399 3.079 PNT .405 .091 19.731 1 .000 1.499 1.254 1.793 PENK .368 .088 17.416 1 .000 .692 .582 .823

(63) Highest vs. lowest quartile PNT indicated a 2.56 fold risk for breast cancer development and Pro Enkephalin on top of PNT lowest vs highest quartile (rev=reversed quartiles Q1=Q4, Q2=Q3, Q3=Q2, Q4=Q1)) an independent 3.6 fold risk (table 9).

(64) Combining highest quartile of PNT and lowest Pro-Enkephalin quartile vs. lowest PNT- and highest Pro-Enkephalin quartile showed a combined risk of 6.17 (see FIG. 3).

(65) Table 9: combined analysis of PNT and PENK for breast cancer prediction.

(66) TABLE-US-00012 TABLE 9 Variables in the Equation 95.0% CI B SE Wald df Sig. Exp(B) Lower AGE .022 .018 1.468 1 .226 .978 .943 current_smoker0 .391 .200 3.808 1 .051 1.478 .998 hrt_curr .652 .195 11.145 1 .001 1.920 1.309 BMI_B .012 .025 .247 1 .619 1.012 .964 GFR_CG_BSAcorr .012 .008 2.279 1 .131 .988 .972 NLN_PNT 13.898 3 .003 NLN_PNT(1) .353 .301 1.378 1 .241 1.424 .789 NLN_PNT(2) .604 .286 4.452 1 .035 1.830 1.044 NLN_PNT(3) .942 .269 12.260 1 .000 2.566 1.514 Q_PENK_rev 23.361 3 .000 Q_PENK_rev(1) .410 .331 1.534 1 .215 1.507 .787 Q_PENK_rev(2) .979 .305 10.299 1 .001 2.663 1.464 Q_PENK_rev(3) 1.284 .300 18.315 1 .000 3.610 2.005

(67) FIG. 4: Illustration example of combined analysis of Pro-Enkephalin for breast cancer prediction:

(68) We combined the women with lowest Pro-Enkephalin (1.sup.st) quartile and highest (4.sup.th) Pro-Neurotensin quartile (group 3). Within that high risk group about 19.02% of women developed breast cancer within the following 15 years.

(69) Group 2 is a combination of women with 3.sup.rd quartile of Pro-Neurotensin and 2.sup.nd quartile of Pro-Enkephalin plus 2.sup.nd quartile of Pro-Neurotensin and 3th quartile of Pro-Enkephalin. Within that medium risk group about 7.48% of women developed breast cancer within the following 15 years.

(70) Group 1 is a combination of women with 1.sup.st quartile of Pro-Neurotensin and 4.sup.th quartile of Pro-Enkephalin. Within that low risk group about 3.08% of women developed breast cancer within the following 15 years. The Hazard risk between group 1 and group 3 is about 6.17.

(71) Lung Cancer

(72) Pro-Enkephalin also predicts lung cancer in females.

(73) 40 women developed lung cancer during the observation period. Pro-Enkephalin is not different in smoking and not smoking women (p=0.44). As expected, smoking is a strong risk prediction marker for lung cancer (p<0.0001). Surprisingly, although smoking is part of the equation, low Pro-Enkephalin indicated a 3.2 fold risk of developing lung cancer (table 10a and 10b).

(74) Table 10 a and 10 b: PENK (SEQ ID NO. 1) in the prediction of lung cancer in females. The women were grouped in tertiles (see table 10 a) and then analyzed for lung cancer development (see table 10 b). rev=highest tertile (tertile 3), rev (1)=tertile 2 and rev(2)=lowest tertile (tertile 1).

(75) TABLE-US-00013 TABLE 10 a PENK [pmol/L] Percentile Group of PENKpmolL Median Minimum Maximum 1 37,80000 9,000 42,800 2 47,20000 42,900 51,300 3 58,30000 51,400 518,100 Total 47,25000 9,000 518,100

(76) TABLE-US-00014 TABLE 10 b Variables in the Equation B SE Wald df Sig. Exp(B) AGE .045 .040 1.251 1 .263 1.046 current_smoker0 1.897 .427 19.761 1 .000 6.667 BMI_B .034 .063 .287 1 .592 .967 GFR_CG_BSAcorr .024 .019 1.592 1 .207 .976 T_PENK_females_rev 6.698 2 .035 T_PENK_females_rev(1) .208 .580 .128 1 .721 1.231 T_PENK_females_rev(2) 1.168 .511 5.220 1 .022 3.214

FIGURE DESCRIPTION

(77) FIG. 1: shows a typical Pro-Enkephalin dose/signal curve. Standard curve Pro-Enkephalin.

(78) FIG. 2: frequency distribution of Pro-Enkephalin in the females population:

(79) FIG. 3: Kaplan Meier graphs, illustrating the cumulative breast cancer diagnosis in women quartile (Q) 1 (below 40.4 pmol/l) quartile 2 (40.4-47.1 pmol/l), quartile 3 (47.2-54.1 pmol/l), quartile 4 (above 54.1 pmol/l). Decreased PENK indicates a long term increased risk of breast cancer development. Since any women with cancer history at day of baseline (blood sampling) were excluded, Pro-Enkephalin is highly predictive for future breast cancer development. Over all, women from Q 1 have a 3.6 times higher risk to develop breast cancer than women from Q 4.

(80) FIG. 4: Illustration example of combined analysis of Pro-Enkephalin for breast cancer prediction: