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

1-20. (canceled)

21. A method for predicting a risk of getting breast cancer or lung cancer in a female subject that does not suffer from breast cancer or lung cancer comprising: measuring the level of Pro-Enkephalin (SEQ ID No. 1), or one or more fragments thereof having at least 5 amino acids, in a sample of bodily fluid obtained from said female subject using an immunoassay that has antibodies or fragments of antibodies that bind to Pro-Enkephalin (SEQ ID No. 1) or said one or more fragments thereof; and correlating said level of Pro-Enkephalin (SEQ ID No. 1), or of said one or more fragments thereof, with risk for getting breast cancer or lung cancer, wherein a reduced level of Pro-Enkephalin (SEQ ID No. 1), or of said one or more fragments thereof is predictive for an enhanced risk of getting breast cancer or lung cancer in comparison to a normal female subject; wherein said one or more fragments thereof is selected from SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, wherein the reduced level of Pro-Enkephalin or fragments thereof is a level below 100 pmol/l, 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 (SEQ ID No. 1) or said one or more fragments thereof whereby Pro-Enkephalin (SEQ ID No. 1) or said one or more 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 (SEQ ID No. 1) or said one or more 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.

22. The method according to claim 21, wherein said cancer is-lung cancer.

23. The method according to claim 21, wherein said female subject has never had a history of diagnosis of breast cancer or lung cancer at the time said sample of bodily fluid is taken from said female subject.

24. The method according to claim 21, wherein said female subject has had a history of diagnosis of breast cancer and has been cured at the time said sample of bodily fluid is taken from said female subject and the risk of reoccurrence of getting breast cancer is determined.

25. The method according to claim 21, wherein at the time said sample of bodily fluid is taken from said female subject, said female subject has been diagnosed as having a cardiovascular disease or diabetes.

26. The method according to claim 21, further comprising determining at least one clinical parameter selected from: age, presence of diabetes mellitus, and currently smoking.

27. The method according to claim 21, wherein said method is performed more than once in order to monitor the risk of getting breast cancer in said female subject.

28. The method according to claim 21, wherein said a method is performed to monitor the response of said female subject to preventive and/or therapeutic measures taken following an assessment of the risk of getting breast cancer or lung cancer.

29. The method according to claim 21, wherein said a method is performed in order to stratify female subjects into risk groups.

30. The method according to claim 21, wherein said method further comprises: measuring the level of Pro-Neurotensin 1-117 (SEQ ID No. 17) in a sample of bodily fluid obtained from said female subject; and correlating said level of Pro-Neurotensin 1-117 (SEQ ID No. 17) with risk for getting breast cancer or lung cancer, wherein an increased level of Pro-Neurotensin 1-117 (SEQ ID No. 17) is predictive for an enhanced risk of getting breast cancer or lung cancer in comparison to a normal female subject.

31. The method according to claim 21, wherein said reduced level of Pro-Enkephalin (SEQ ID No. 1) or of said one or more fragments thereof is a level below 50 pmol/l.

32. The method according to claim 30, wherein said increased level of Pro-Neurotensin 1-117 (SEQ ID No. 17) is a level above 78 pmol/l.

33. The method according to claim 21, wherein said bodily fluid is blood, plasma or serum.

34. The method according to claim 21, wherein the level of a fragment of Pro-Enkephalin (SEQ ID No. 1) is measured and said fragment is MR- Pro-Enkephalin (SEQ ID No. 6).

35. A kit for determining Pro-Enkephalin (SEQ ID No. 1) and/or one or more fragments thereof in a sample comprising a first and second binder, wherein the first binder binds to a region of Pro-Enkephalin (SEQ ID No. 1) that is within amino acid sequence 133-140 (LKELLETG, SEQ ID NO. 22), and the second binder binds to a region of Pro-Enkephalin (SEQ ID No. 1) that is within amino acid sequence 152-159 (SDNEEEVS, SEQ ID NO. 23), wherein each of said regions comprises at least 4 or 5 amino acids.

36. The kit according to claim 35, wherein said kit is sufficiently sensitive to quantify the level of Pro-Enkephalin (SEQ ID No. 1) or said one or more fragments thereof of healthy subjects and has an analytical assay sensitivity < 15 pmol/L.

37. A method for predicting a risk of getting cancer in a female subject that does not suffer from breast cancer or lung cancer, and optionally for stratification of female subjects into risk groups, said method comprising: measuring the level of Pro-Enkephalin (SEQ ID No. 1), or of one or more fragments thereof, in a sample of bodily fluid obtained from a female subject using said kit according to claim 35; correlating said level of Pro-Enkephalin, or of one or more fragments thereof, with risk for getting breast cancer or lung cancer, wherein a reduced level of Pro-Enkephalin (SEQ ID No. 1), or of said one or more fragments thereof is predictive for an enhanced risk of getting breast cancer or lung cancer in comparison to a normal female subject; and optionally using said method to stratify female subjects into risk groups, wherein the reduced level of Pro-Enkephalin or fragments thereof is a level below 100 pmol/l.

38. The method according to claim 37, wherein said method is used for stratification of female subjects into a group that should obtain opioid growth factor (OGF) therapy.

39. The method according to claim 21, wherein said cancer is breast cancer.

40. The method according to claim 30, wherein said cancer is breast cancer.

41. The method according to claim 21, wherein said correlating is performed by: (a) comparing the level of Pro-Enkephalin (SEQ ID No. 1) or of said one or more fragments thereof in said sample with the median of the level of Pro-Enkephalin (SEQ ID No. 1) or of said one or more fragments thereof in an ensemble of pre-determined samples in a population of “healthy” or “apparently healthy” subjects, (b) comparing the level of Pro-Enkephalin (SEQ ID No. 1) or of said one or more fragments thereof in said sample with a quantile of the level of Pro-Enkephalin (SEQ ID No. 1) or of said one or more fragments thereof in 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 (SEQ ID No. 1) or of said one or more fragments thereof in said sample.

42. A method according to claim 21, 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 (SEQ ID No. 1), 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 (SEQ ID No. 1).

43. A method according to claim 30, 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 (SEQ ID No. 1), 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 (SEQ ID No. 1).

44. A method according to claim 30, wherein said measuring of the level of Pro-Neurotensin 1-117 (SEQ ID No. 17) comprises: a) bringing said sample of bodily fluid into contact with a solid phase comprising a bound third antibody or third antibody fragment that binds to Pro-Neurotensin 1-117 (SEQ ID No. 17) whereby Pro-Neurotensin 1-117 (SEQ ID No. 17) 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, c) measuring the level of Pro-Neurotensin 1-117 (SEQ ID No. 17) in said sample by measuring the amount of labelled fourth antibody or fourth antibody fragment bound to the complex on said solid phase.

45. A method according to claim 44, 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 1-117 (SEQ ID No. 17), 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 1-117 (SEQ ID No. 17).

46. A method according to claim 45, 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 (SEQ ID No. 1), 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 (SEQ ID No. 1).

47. The method according to claim 21, wherein said measuring of the level of Pro-Enkephalin (SEQ ID No. 1), or one or more fragments thereof of at least 5 amino acids, is performed using a kit for determining Pro-Enkephalin (SEQ ID No. 1) and/or one or more fragments thereof in a sample comprising a first and second binder, wherein the first binder binds to a region of Pro-Enkephalin (SEQ ID No. 1) that is within amino acid sequence 133-140 (LKELLETG, SEQ ID NO. 22), and the second binder binds to a region of Pro-Enkephalin (SEQ ID No. 1) that is within amino acid sequence 152-159 (SDNEEEVS, SEQ ID NO. 23), wherein each of said regions comprises at least 4 or 5 amino acids.

48. The method according to claim 21, wherein said antibodies or fragments of antibodies that bind to Pro-Enkephalin or said one or more fragments thereof are prepared by: immunizing a mouse with a peptide-BSA-conjugate, wherein BSA is bovine serum albumin and said peptide is an amino acid sequence selected from SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11 in which the amino acid sequence is provided with an additional N-terminal cysteine residue for conjugation to bovine serum albumin; fusing spenocytes obtained from the immunized mouse with cells of a myeloma cell line; and producing said antibodies or fragments of antibodies from the resultant fused cells.

49. A method comprising: obtaining a sample of bodily fluid from a female subject that does not suffer from breast cancer or lung cancer; combining said sample with a binder that binds to the amino acid sequence of SEQ ID No. 6; measuring the level of bound binder within said sample to be below 100 pmol/1; and subjecting said female subject to measures for preventing breast cancer or lung cancer, or subjecting said female subject to measures for treating breast cancer or lung cancer.

50. A method comprising: subjecting a female subject to measures for preventing breast cancer or lung cancer, or subjecting a female subject to measures for treating breast cancer or lung cancer, wherein said female subject that does not suffer from breast cancer or lung cancer and, prior to subjecting said female subject to measures for preventing or treating breast cancer or lung cancer, a sample of bodily fluid obtained from said female subject has been combined with a binder that binds to the amino acid sequence of SEQ ID No. 6 and the level of bound binder within said sample has been determined to be below 100 pmol/l.

Description

FIGURE DESCRIPTION

[0126] FIG. 1: shows a typical Pro-Enkephalin dose/ signal curve. Standard curve Pro-Enkephalin.

[0127] FIG. 2: frequence distribution of Pro-Enkephalin in the females population:

[0128] 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.

[0129] FIG. 4: Illustration example of combined analysis of Pro-Enkephalin for breast cancer prediction:

[0018] The term “subject” as used herein refers to a living human or non-human organism. Preferably herein the subject is a human subject.

[0019] The term “reduced level” means a level below a certain threshold level.

[0020] A bodily fluid may be selected from the group comprising blood, serum, plasma, urine, cerebro spinal liquid (csf), and saliva.

[0021] In one embodiment of the invention said female subject has never had a diagnosed cancer at the time the sample of bodily fluid is taken from said female subject.

[0022] In another embodiment said female subject has been diagnosed before with having cancer and has been cured at the time the sample of bodily fluid is taken from said female subject and the risk of reoccurrence of getting cancer is determined or alternatively the re-occurrence of cancer is predicted.

[0023] Pro-Enkephalin has the following sequence:

[0024] SEQ ID NO. 1 (Pro-Enkephalin (1-243)

TABLE-US-00001 ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTL RENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMK KDAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQL EDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPE MEKRYGGF MRF

[0025] Fragments of Pro-Enkephalin that may be determined in a bodily fluid may be e.g. selected from the group of the following fragments:

[0026] SEQ ID NO. 2 (Synenkephalin, Pro-Enkephalin 1-73)

TABLE-US-00002 ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTL RENSKPEESHLLA

[0027] SEQ ID NO. 3 (Met-Enkephalin)

TABLE-US-00003 YGGFM

[0028] SEQ ID NO. 4 (Leu-Enkephalin)

TABLE-US-00004 YGGFL

[0029] SEQ ID NO. 5 (ProEnkephalin 90-109)

TABLE-US-00005 MDELYPMEPEEEANGSEILA

[0030] SEQ ID NO. 6 (Pro Enkephalin 119-159, Mid regional Pro-Enkephalin-fragment, MRPENK)

TABLE-US-00006 DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS

[0031] SEQ ID NO. 7 (Met-Enkephalin-Arg-Gly-Leu)

TABLE-US-00007 YGGFMRGL

[0032] SEQ ID NO. 8 (Pro-Enkephalin 172-183)

TABLE-US-00008 SPQLEDEAKELQ

[0033] SEQ ID NO. 9 (Pro-Enkephalin 193-203)

TABLE-US-00009 VGRPEWWMDYQ

[0034] SEQ ID NO. 10 (Pro-Enkephalin 213-234)

TABLE-US-00010 FAEALPSDEEGESYSKEVPEME

[0035] SEQ ID NO. 11 (Pro-Enkephalin 213-241)

TABLE-US-00011 FAEALPSDEEGESYSKEVPEMEKRYGGF M

[0036] SEQ ID NO. 12 (Met-Enkephalin-Arg-Phe)

TABLE-US-00012 YGGFMRF

[0037] Determining the level of Pro-Enkephalin including Leu-Enkephalin and Met-Enkephalin or fragments thereof may mean that the immunoreactivity towards Pro-Enkephalin or fragments thereof including Leu-Enkephalin and Met-Enkephalin is determined. A binder used for determination of Pro-Enkephalin including Leu-Enkephalin and Met-Enkephalin or fragments thereof depending of the region of binding may bind to more than one of the above displayed molecules. This is clear to a person skilled in the art.

[0038] Thus, according to the present invention the level of immunoreactive analyte by using at least one binder that binds to a region within the amino acid sequence of any of the above peptide and peptide fragments, (i.e. Pro-Enkephalin (PENK) and fragments according to any of the sequences 1 to 12), is determined in a bodily fluid obtained from said subject; and correlated to the specific embodiments of clinical relevance.

[0039] In a more specific embodiment of the method according to the present invention the level of MRPENK is determined (SEQ ID NO. 6: Pro-Enkephalin 119-159, Mid regional Pro-Enkephalin-fragment, MRPENK). In a more specific embodiment the level of immunoreactive analyte by using at least one binder that binds to MR-PENK is determined and is correlated to the specific embodiments of clinical relevance according to the invention.

[0040] Determining the level of Pro-Enkephalin or fragments thereof including Leu-Enkephalin and Met-Enkephalin or fragments thereof may mean that the immunoreactivity towards Pro-Enkephalin or fragments thereof including Leu-Enkephalin and Met-Enkephalin is determined. A binder used for determination of Pro-Enkephalin including Leu-Enkephalin and Met-Enkephalin or fragments thereof depending of the region of binding may bind to more than one of the above displayed molecules. This is clear to a person skilled in the art. In another embodiment of the invention the fragment is not Leu-Enkephalin or Met-Enkephalin, In another embodiment of the invention the immunoreactivity towards Pro-Enkephalin or fragments thereof not including Leu-Enkephalin and Met-Enkephalin is determined.

[0041] In a more specific embodiment of the method according to the present invention the level of MRPENK. (SEQ ID NO. 6 (Pro Enkephalin 119-159, Mid regional Pro-Enkephalin-fragment, MRPENK, DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS) is determined.

[0042] Alternatively the level of any of the above analytes may be determined by other analytical methods e.g. mass spectroscopy.

[0043] In a specific embodiment the level of Pro-Enkephalin or fragments thereof are measured with an immunoassay using antibodies or fragments of antibodies binding to Pro-Enkephalin or fragments thereof. An immunoassay that may be useful for determining the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids may comprise the steps as outlined in Example 2. All thresholds and values have to be seen in correlation to the test and the calibration used according to Example 2. A person skilled in the art may know that the absolute value of a threshold might be influenced by the calibration used. This means that all values and thresholds given herein are to be understood in context of the calibration used in herein (Example 2). According to the invention the diagnostic binder to Pro-Enkephalin is selected from the group consisting of antibodies e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and/or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized with the CH3 domain; bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid of a heterologous domain, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab′)2-fragments, scFv-fragments, multimerized multivalent or/and multispecific scFv-fragments, bivalent and/or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulines.

[0044] In a specific embodiment the level of Pro-Enkephalin or fragments thereof are measured with an assay using binders selected from the group comprising aptamers, non-Ig scaffolds as described in greater detail below binding to Pro-Enkephalin or fragments thereof.

[0045] Binder that may be used for determining the level of Pro-Enkephalin or fragments thereof exhibit an affinity constant to Pro-Enkephalin of at least 10.sup.7 M.sup.-1, preferred 10.sup.8 M.sup.-1, preferred affinity constant is greater than 10.sup.9 M.sup.-1, most preferred greater than 10.sup.10 M.sup.-1. A person skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention. Binding affinity may be determined using the Biacore method, offered as service analysis e.g. at Biaffin, Kassel, Germany (http://www.biaffin.com/de/).

[0046] A human Pro-Enkephalin-control sample is available by ICI-Diagnostics, Berlin, Germany http://www.ici-diagnostics.com/. The assay may also be calibrated by synthetic (for our experiments we used synthetic MRPENK, SEQ ID NO. 6) or recombinant Pro-Enkephalin or fragments thereof.

[0047] The threshold for determining the risk of getting breast cancer in a female subject or diagnosing breast cancer in a female subject according to the methods of the present invention is below 100 pmol/l PENK, preferred below 50 pmol/l, more preferred below 40.4 pmol/l. In a specific embodiment said threshold is about 40.4 pmol/l. These thresholds are related to the above mentioned calibration method. A PENK value below said threshold means that the subject has an enhanced risk of getting cancer or has already cancer.

[0048] In one embodiment of the invention said method is performed more than once in order to monitor the risk of getting breast cancer in a female subject or in order to monitor the course of treatment. In one specific embodiment said monitoring is performed in order to evaluate the response of said female subject to preventive and/or therapeutic measures taken.

[0049] In one embodiment of the invention the method is used in order to stratify said female subjects into risk groups.

[0050] Subject of the present invention is also a method for predicting the risk of getting cancer in a female or identifying a female subject having an enhanced risk for getting cancer according to any of the preceding embodiments, wherein the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said female subject either alone or in conjunction with other prognostically useful laboratory or clinical parameters is used for the prediction of a subject’s risk for getting an adverse event by a method which may be selected from the following alternatives: [0051] Comparison with the median of the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said female subject in an ensemble of predetermined samples in a population of “healthy” or “apparently healthy” subjects, [0052] Comparison with a quantile of the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said female subject in an ensemble of predetermined samples in a population of “healthy” or “apparently healthy” subjects, [0053] Calculation based on Cox Proportional Hazards analysis or by using Risk index calculations such as the NRI (Net Reclassification Index) or the IDI (Integrated Discrimination Index).

[0054] In one embodiment of the invention subject of the present invention is also a method for predicting the risk of getting cancer in a female or identifying a female subject having an enhanced risk for getting cancer according to any of the preceding embodiments, wherein the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said female subject either alone or in conjunction with other prognostically useful biomarker. Such a useful biomarker may be Pro-Neurotensin and fragments thereof of at least 5 amino acids.

[0055] In a more specific embodiment of the method according to the present invention the level of Pro-Neurotensin 1-117 is determined in addition to the determination of Pro-Enkephalin an fragments thereof.

[0056] Thus, subject matter of the present invention is also 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: [0057] 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 [0058] determining the level of Pro-Neurotensin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said female subject; and [0059] correlating said level of Pro-Enkephalin or fragments thereof and Pro-Neurotensin or fragments thereof of at least 5 amino acids with a risk for getting cancer, wherein an reduced level of Pro-Enkephalin is predictive for an enhanced risk of getting cancer or alternatively diagnosing cancer wherein an reduced level is correlated with the diagnosis of cancer and wherein an increased level of Pro-Neurotensin is predictive for an enhanced risk of getting cancer or alternatively diagnosing cancer wherein an increased level is correlated with the diagnosis of cancer.

[0060] SEQ ID NO. 13 (Pro-Neurotensin 1-147)

TABLE-US-00013 SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVIKRK IPYILKRQLY ENKPRRPYIL KRDSYYY

[0061] SEQ ID NO. 14 (Pro-Neurotensin 1-125 (large neuromedin N))

TABLE-US-00014 SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVI KR KIPYIL

[0062] SEQ ID NO. 15 (neuromedin N)

TABLE-US-00015 KIPYIL

[0063] SEQ ID NO. 16 (neurotensin)

TABLE-US-00016 pyroQLYENKPRRP YIL

[0064] SEQ ID NO. 17 (Pro-Neurotensin 1-117)

TABLE-US-00017 SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVI

[0065] SEQ ID NO. 18 (Pro-Neurotensin 1-132)

TABLE-US-00018 SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVIKRK IPYILKRQLY EN

[0066] SEQ ID NO. 19 (Pro-Neurotensin 120-140)

TABLE-US-00019 KIPYILKRQL YENKPRRPYI L

[0067] SEQ ID NO. 20 (Pro-Neurotensin 120-147)

TABLE-US-00020 KIPYILKRQL YENKPRRPYIL KRDSYYY

[0068] SEQ ID NO. 21 (Pro-Neurotensin 128-147)

TABLE-US-00021 QLYENKPRRP YILKRDSYYY

[0069] In a specific embodiment the level of Pro-Neurotensin is measured with an immunoassay. More specifically an immunoassay is used as described in Ernst et al. (Peptides (2006), (27) 1787-1793). An immunoassay that may be useful for determining the level of Pro-Neurotensin or fragments thereof of at least 5 amino acids may comprise the steps as outlined in Example 2. All thresholds and values have to be seen in correlation to the test and the calibration used according to Example 2. A person skilled in the art may know that the absolute value of a threshold might be influenced by the calibration used. This means that all values and thresholds given herein are to be understood in context of the calibration used in herein (Example 2). A human Pro-Neurotensin-calibrator is available by ICI-Diagnostics, Berlin, Germany. Alternatively, the assay may also be calibrated by synthetic or recombinant P—NT 1-117 or fragments thereof (see also Ernst et al, 2006).

[0070] Binder that may be used for determining the level of Pro-Neurotensin or fragments thereof exhibit an affinity constant to Pro-Neurotensin of at least 10.sup.7 M.sup.-1, preferred 10.sup.8 M.sup.-1, preferred affinity constant is greater than 10.sup.9 M.sup.-1, most preferred greater than 10.sup.10 M.sup.-1. A person skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention. Binding affinity may be determined using the Biacore method, offered as service analysis e.g. at Biaffin, Kassel, Germany (http://www.biaffin.com/de/).The threshold for determining the risk of getting breast cancer in a female subject or diagnosing breast cancer in a female subject according to the methods of the present invention is above 78 pmol/l PNT, preferred 100 pmol/l, more preferred 150 pmol/l. In a specific embodiment said threshold is about 100 pmol/l. These thresholds are related to the above mentioned calibration method. A P-NT value above said threshold means that the subject has an enhanced risk of getting cancer or has already cancer.

[0071] In one embodiment of the invention said method is performed more than once in order to monitor the risk of getting breast cancer in a female subject or in order to monitor the course of treatment. In one specific embodiment said monitoring is performed in order to evaluate the response of said female subject to preventive and/or therapeutic measures taken.

[0072] In one embodiment of the invention the method is used in order to stratify said female subjects into risk groups.

[0073] In one embodiment of the invention the cancer is selected from the group comprising breast cancer, and lung cancer. Subject matter of the invention is further an assay for determining Pro-Enkephalin and Pro-Enkephalin fragments in a sample comprising two binders that bind to two different regions within the region of Pro-Enkephalin that is aminoacid 133-140 (LKELLETG, SEQ ID NO. 22) and aminoacid 152-159 (SDNEEEVS, SEQ ID NO. 23) wherein each of said regions comprises at least 4 or 5 amino acids.

[0074] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention the assay sensitivity of said assay is able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is < 15 pmol/, preferably < 10 pmol/l and more preferably L < 6 pmol/L.

[0075] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention said binder exhibits an binding affinity to its binding partner of at least 10.sup.7 M.sup.-1, preferred 10.sup.8 M.sup.-1, preferred affinity constant is lower than 10.sup.9 M.sup.-1, most preferred lower than 10.sup.10 M.sup.-1. A person skilled .sub.[K1] in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention binding affinity may be determined as described above.

[0076] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention such assay is a sandwich assay, preferably a fully automated assay. It may be an ELISA fully automated or manual. It may be a so-called POC-test (point -of-care). Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®. Examples of test formats are provided above.

[0077] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention at least one of said two binders is labeled in order to be detected. Examples of labels are provided above.

[0078] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention at least one of said two binders is bound to a solid phase. Examples of solid phases are provided above.

[0079] In one embodiment of the assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention said label is selected from the group comprising chemiluminescent label, enzyme label, fluorescence label, radioiodine label.

[0080] A further subject of the present invention is a kit comprising an assay according to the present invention wherein the components of said assay may be comprised in one or more container.

EXAMPLES

Example 1

Development of Antibodies

Peptides

[0081] Peptides were synthesized (JPT Technologies, Berlin, Germany).

Peptides/Conjugates for Immunization

[0082] 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.

TABLE-US-00022 Peptide for immunization Pro-Enkephalin sequence (C)DAEEDD (SEQ ID NO: 26) 119-125 (C)EEDDSLANSSDLLK (SEQ ID NO: 27) 121-134 (C)LKELLETG (SEQ ID NO: 28) 133-140 (C)TGDNRERSHHQDGSDNE (SEQ ID NO: 29) 139-155 (C)SDNEEEVS (SEQ ID NO: 30) 152-159

[0083] The antibodies were generated according to the following method:

[0084] A BALB/c mouse was immunized with 100 .Math.g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 .Math.l complete Freund’s adjuvant) and 50 .Math.g at day 21 and 28 (in 100 .Math.l incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received 50 .Math.g of the conjugate dissolved in 100 .Math.l saline, given as one intraperitonal and one intravenous injection.

[0085] 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.

[0086] 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.

[0087] (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)).

Monoclonal Antibody Production

[0088] 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.

Labelling and Coating of Antibodies

[0089] All antibodies were labelled with acridinium ester according the following procedure:

[0090] Labelled compound (tracer): 100 .Math.g (100 .Math.l) antibody (1 mg/ml in PBS, pH 7.4, was mixed with 10 .Math.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 .Math.l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Solid Phase Antibody (Coated Antibody)

[0091] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 .Math.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.

Antibody Specificity

[0092] The crossreactivities of the different antibodies are listed in table 2.

TABLE-US-00023 Peptide for immunization Pre-Pro-Enkephalin-sequence Antibody name (C)DAEEDD (SEQ ID NO: 26) 119-125 NT-MRPENK (C)EEDDSLANSSDLLK (SEQ ID NO: 27) 121-134 NM-MRPENK (C)LKELLETG (SEQ ID NO: 28) 133-140 MR-MRPENK (C)TGDNRERSHHQDGSDNE (SEQ ID NO: 29) 139-155 MC-MRPENK (C)SDNEEEVS (SEQ ID NO: 30) 152-159 CT-MRPENK

[0093] Antibody cross-reactivities were determined as follows:

[0094] 1ug peptide in 300 .Math.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 .Math.l in PBS, pH 7.4, 800.000 RLU/ 300 .Math.l) an incubated for 2 h at room temperature, After washing 5 times (each 1 ml of washing solution (20 mmol/l PBS, pH 7.4, 0.1 % Triton X 100), the remaining luminescence (labelled antibody) was quantified using the AutoLumat LB 953. MRPENK-peptide was used as reference substance (100%).

TABLE-US-00024 antibody peptide DAEEDD (SEQ ID NO: 31) EEDDSLANSSD LLK (SEQ ID NO: 32) LKELLETG (SEQ ID NO: 22) TGDNRERSH HQDGSDNE (SEQ ID NO: 33) SDNEEEVS (SEQ ID NO: 23) MRPENK (SEQ ID NO. 6) NT-MRPENK 121 10 <1 <1 <1 100 NM-MRPENK <1 98 <1 <1 <1 100 MR-MRPENK <1 <1 105 <1 <1 100 MC-MRPENK <1 <1 <1 115 <1 100 CT-MRPENK <1 <1 <1 <1 95 100

[0095] All antibodies bound the MRPENK peptide, compareable to the peptides which were used for immunization. Except for NT-MRPENK-antibody (10% cross reaction with EEDDSLANSSDLLK (SEQ ID NO: 32)) no antibody showed a cross reaction with MR-PENK peptides not used for immunization of the antibody.

Pro-Enkephalin Immunoassay

[0096] 50 .Math.l of sample (or calibrator) was pipetted into coated tubes, after adding labeled antibody (200 ul), 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.

TABLE-US-00025 Solid phase antibody NT-MRPENK NM-MRPENK MR-MRPENK MC-MRPENK CT-MRPENK Labelled antibody NT-MRPENK / 27 212 232 <1 NM-MRPENK 36 / 451 487 <1 MR-MRPENK 175 306 / 536 1050 MC-MRPENK 329 577 542 / <1 CT-MRPENK <1 615 1117 516 /

Calibration

[0097] The assay was calibrated, using dilutions of synthetic MRPENK, diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 .Math.M Amastatin, 100 .Math.M Leupeptin, pH 8.0. Pro-Enkephalin control plasma is available at ICI-diagnostics, Berlin, Germany.

[0098] FIG. 1 shows a typical Pro-Enkephalin dose/ signal curve. Standard curve Pro-Enkephalin.

[0099] The assay sensitivity was 20 determinations of 0-calibrator (no addition of MRPENK) + 2SD) 5.5 pmol/L.

Population Study

Methods

[0100] We measured Pro-Enkephalin in fasting plasma from 2559 female participants of the population based Malmö Diet and Cancer Study baseline exam in 1991-1994 (age 58 ± 6 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 Malmö 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).

[0101] Clinical characteristics of females in the study

TABLE-US-00026 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 x 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 0.58404 High density lipoprotein (mmol/l) 2559 1.5123 0.36949 Low density lipoprotein (mmol/l) 2559 4.2016 1.04762 P-INSULIN 2512 7.223 5.4223

[0102] FIG. 2: frequence distribution of Pro Enkephalin in the females population:

[0103] 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).

PENK and Prediction of Breast Cancer

[0104] 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).

TABLE-US-00027 Variables in the Equation° B SE Wald df Sig. Exp(B) 95.0% Cl for Exp(B) Lower Upper AGE 0.007 0.016 0.228 1 0.633 1.007 0.977 1.039 SEX 0.sup.a BMI_B 0.026 0.025 1.139 1 0.286 1.027 0.978 1.077 DM_B -0.242 0.407 0.352 1 0.553 0.785 0.354 1.744 HDL_B 0.044 0.252 0.031 1 0.860 1.045 0.638 1.714 LDL_B -0.001 0.090 0.000 1 0.988 0.999 0.837 1.191 current_smoker 0.330 0.195 2.886 1 0.089 1.392 0.950 2.037 HER_CANCER_0 0.034 0.176 0.038 1 0.846 1.035 0.733 1.461 LNINS -0.288 0.197 2.127 1 0.145 0.750 0.509 1.104 ZscoreLNPENK_ females_noCa -0.337 0.082 16.858 1 0.000 0.714 0.608 0.839

TABLE-US-00028 BREAST CANCER HR per 1 SD P-value Quartile 4 Quartile 3 Quartile 2 Quartile 1 P for trend Women (2140 / 135) 1.40 (13-1.6) <0.001 1.0 (ref) 1.50 (0.81-2.1) 2.7(1.7-3.4) 3.6 (2.7-4.9) <0.001 Multivariate Cox proportional Hazards models for baseline Pro-Enkephalin versus incidence of breast cancer.

[0105] 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.

COMBINATION PRO ENKEPHALIN AND PRO NEUROTENSIN

[0106] Since increasing Pro-Neurotensin recently was shown to be highly predictive for breast cancer, we combined both biomarkers for breast cancer prediction.

EXAMPLES

Pro-Neurotensin Assay

[0107] Antibodies were generated as described above. The antibody for labelling (LA) was generated against P—NT 119 (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)).

Immunoassay for the Quantification of Human Pro-Neurotensin

[0108] The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.

[0109] Labelled compound (tracer): 100 .Math.g (100 .Math.l) LA (1 mg/ml in PBS, pH 7.4, was mixed with 10 .Math.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 .Math.l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0110] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with SPA (1.5 .Math.g SPA/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 vakuum dried.

Calibration

[0111] 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).

[0112] 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 1117 or fragments thereof (see also Ernst et al., 2006).

ProNT Immunoassay

[0113] 50 .Math.l of sample (or calibrator) was pipetted into SPA coated tubes, after adding labeleld 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.

[0114] Combined analysis of Pro-Enkephalin and PNT in the female population: 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.

[0115] 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 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).

TABLE-US-00029 combined analysis of PNT and PENK for breast cancer prediction Variables in the Equation B SE Wald df Sig. Exp(B) 95.0% Cl for Exp(B) Lower Upper AGE -0.003 0.019 0.020 1 0.888 0.997 0.960 1.036 current_smoker0 0.434 0.204 4.505 1 0.034 1.543 1.034 2.304 BMI_B 0.001 0.027 0.001 1 0.979 1.001 0.948 1.056 GFR_CG_BSAcorr -0.005 0.008 0.357 1 0.550 0.995 0.979 1.011 hrt_curr 0.730 0.201 13.146 1 0.000 2.075 1.399 3.079 PNT 0.405 0.091 19.731 1 0.000 1.499 1.254 1.793 PENK -0.368 0.088 17.416 1 0.000 0.692 0.582 0.823

[0116] 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).

[0117] 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).

[0118] Table 9: combined analysis of PNT and PENK for breast cancer prediction.

TABLE-US-00030 Variables in the Equation 95.0% Cl SE Wald df Sig. Exp(B) Lower B AGE -0.022 0.018 1.468 1 0.226 0.976 0.943 current_smoker0 0.391 0.200 3.808 1.051 1.478 0.998 Nt_curr 0.652 0.195 11.145 1 0.001 1.920 1.309 BMI_B 0.012 0.025 0.247 1 0.619 1.012 0.964 GFR_CG_BSAcurr -0.012 0.008 2.279 1 0.131 0.968 0.972 NLN_PNT 13.898 3 0.003 NLN_PNT(1) 0.353 0.301 1.378 1 0.241 1.424 0.789 NLN_PNT(2) 0.604 0.286 4.452 1 0.035 1.630 1.044 NLN_PNT(3) 0.942 0.269 12.280 1 0.000 2.566 1.514 Q_PENK_rev 23.381 3 0.000 Q_PENK_rev(1) 0.410 0.331 1.534 1 0.215 1.507 0.787 Q_PENK_rev(2) 0.979 0.306 10.299 1 0.001 2.663 1.464 Q_PENK_rev(3) 1.284 0.300 18.315 1 0.000 3.610 2.005

[0119] FIG. 4: Illustration example of combined analysis of Pro-Enkephalin for breast cancer prediction:

[0120] 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.

[0121] Group 2 is a combination of women with 3.sup.nd 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.

[0122] 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.

LUNG CANCER

[0123] Pro-Enkephalin also predicts lung cancer in females.

[0124] 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 10 a and 10 b).

[0125] Table 10 a and 10 b: PENK in the prediction of lung cancer in females. The women were grouped in tertiles (see table 10 a) and than 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).

TABLE-US-00031 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

TABLE-US-00032 Variables in the Equation B SE Wald df Sig. Exp(B) AGE 0.045 0.040 1.251 1 0.263 1.046 current_smokerD 1.897 0.427 19.761 1 0.000 6.667 BMI_B -0.034 0.063 0.287 1 0.592 0.967 GFR_CG_BSAcorr -0.024 0.019 1.592 1 0.207 0.976 T_PENK_females_rev 6.698 2 0.035 T_PENK_females_rev(1) 0.208 0.580 0.128 1 0.721 1.231 T_PENK_females_rev(2) 1.168 0.511 5.220 1 0.022 3.214