HK1212762B - A method for predicting the risk of getting cancer or diagnosing cancer in a female subject - Google Patents

Description

Method for predicting cancer risk or diagnosing cancer in female subjects

The present invention relates to a method for predicting the risk of developing cancer in a female subject who does not have cancer or for diagnosing cancer in a female subject, comprising:

Determining the level of proenkephalin (PENK) or a fragment thereof having at least 5 amino acids (including Leu-enkephalin and Met-enkephalin) in a body fluid obtained from the female subject; and

- correlating the level of said proenkephalin or a fragment thereof with a risk of developing cancer, wherein a reduced level predicts an increased risk of developing cancer, or diagnosing cancer, wherein a reduced level is associated with a diagnosis of cancer.

Met-enkephalin is a 5-amino acid peptide derived from the enkephalin precursor (preproenkephalin), also known as "opioid growth factor" (OGF), which is released along with the proenkephalin fragment. The mature peptide binds to different opioid receptors (Koneru et al, 2009). Enkephalin (OGF) has been found to have multiple physiological functions. In the CNS, it downregulates substance P-related pain signal transduction and acts as a cytokine (Plotnikoff et al, 1997). Peptides related to proenkephalin exhibit antimicrobial effects (Goumon et al, 1998). Proenkephalin and enkephalin exhibit antitumor effects and act as pro-apoptotic agents (Tavish et al, 2007, Donahue et al, 2011, Zagon et al, 2009).

The use of vasoactive peptides for predicting cancer risk in men has been reported by Belting et al., Cancer, Epidemiology, Biomarkers & Prevention. Fasting plasma levels of MR-pro-ANP, MR-pro-ADM, and copeptin were measured in 1768 men and 2293 women from the Malmo Diet and Cancer Study who were cancer-free before a baseline examination between 1991 and 1994. The authors reported no association between the biomarkers and cancer development in women.

The present invention is concerned with the prognostic and diagnostic power of PENK for predicting cancer development and risk of cancer recurrence. To address this question, the stable proenkephalin fragment was measured in fasting plasma in the Swedish prospective cohort study (Malmo Diet and Cancer Study) (Ernst et al, 2006) and the baseline level of this biomarker was correlated with breast cancer incidence during a 15-year follow-up period.

Surprisingly, proenkephalin was shown to be a powerful and highly significant biomarker for females for predicting the risk of developing cancer in female subjects who do not have cancer or for diagnosing cancer in female subjects.

Thus, the subject of the present invention is a method for predicting the risk of developing cancer in a female subject who does not have cancer or for diagnosing cancer in a female subject, comprising:

Determining the level of proenkephalin or a fragment having at least 5 amino acids thereof in a body fluid obtained from the female subject; and

- correlating the level of said proenkephalin or a fragment thereof with the risk of developing cancer, wherein a reduced level predicts an increased risk of developing cancer, or diagnosing cancer, wherein a reduced level is associated with the diagnosis of cancer.

Examples of cancer may be selected from the group consisting of breast cancer, lung cancer, pancreatic cancer and colon cancer.

Throughout the specification it is to be understood that the term fragments of proenkephalin also encompasses Leu-enkephalin and Met-enkephalin.

In a specific embodiment of the present invention, the cancer is breast cancer. In another specific embodiment of the present invention, the cancer is lung cancer.

Therefore, the subject matter of the present invention is to determine the susceptibility of women to cancer, such as breast cancer, lung cancer, etc.

The data obtained in this study also revealed a correlation between the risk of cancer in male subjects and the levels of proenkephalin or fragments thereof having at least 5 amino acids in the body fluids of these male subjects; however, while there was a clear trend toward increased cancer risk in men with similarly decreased PENK levels, this correlation was not statistically significant for the current dataset. Therefore, the methods of the present invention are also valuable for male subjects, but in this study, the effects observed in men were not as strong as in women. This may be primarily due to the lower number of cancers in the male population.

In addition, the data obtained in this study also revealed a correlation between the risk of cancer in female subjects and the level of proenkephalin or a fragment thereof having at least 5 amino acids in the body fluids of the female subjects, wherein the cancer is not lung cancer or breast cancer. However, due to the relatively small number of occurrences in this particular population, the correlation is not statistically significant for the current data set. Although it is not significant, there is still a clear trend. In addition, it is credible that due to the pro-apoptotic effect of known enkephalins (fragments of PENK), current data show that the main association is also present in other cancers. From the perspective of the prior art, it is surprising that proenkephalin or a fragment thereof can predict cancer. From the current data that are statistically highly correlated with breast cancer and lung cancer, it is expected and credible that it can also predict other types of cancer.

As used herein, the term "subject" refers to a living human or non-human organism. Preferably, the subject herein is a human subject.

The term "reduced levels" refers to levels below a certain threshold level.

The body fluid may be selected from the group consisting of: blood, serum, plasma, urine, cerebrospinal fluid (csf) and saliva.

In one embodiment of the present invention, the female subject has never had a diagnosed cancer at the time the body fluid sample is collected from the female subject.

In another embodiment, the female subject has been previously diagnosed with cancer and has been cured at the time the bodily fluid sample is collected from the female subject, and the risk of recurrence of the cancer is determined or recurrence of the cancer is predicted.

Proenkephalin has the following sequence:

SEQ ID NO.1 (Proenkephalin (1-243)

ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMKKDA EEDDSLANSSELLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQLEDEALELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPEMEKRYGGF MRF

The fragments of proenkephalin that can be measured in body fluids can, for example, be selected from the following fragments:

SEQ ID NO.2 (synthetic enkephalin, proenkephalin 1-73)

ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLA

SEQ ID NO.3 (Met-enkephalin)

YGGFM

SEQ ID NO.4 (Leu-enkephalin)

YGGFL

SEQ ID NO.5 (proenkephalin 90-109)

MDELYPMEPEEEANGSEILA

SEQ ID NO.6 (proenkephalin 119-159, middle region proenkephalin-fragment, MRPENK)

DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS

SEQ ID NO.7 (Met-enkephalin-Arg-Gly-Leu)

YGGFMRGL

SEQ ID NO.8 (proenkephalin 172-183)

SPQLEDEAKELQ

SEQ ID NO.9 (proenkephalin 193-203)

VGRPEWWMDYQ

SEQ ID NO.10 (proenkephalin 213-234)

FAEALPSDEEGESYSKEVPEME

SEQ ID NO.11 (proenkephalin 213-241)

FAEALPSKEEGESYSKEVPEMEKRYGGF M

SEQ ID NO.12 (Met-enkephalin-Arg-Phe)

YGGFMRF

Measuring the level of proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin) may refer to measuring immunoreactivity against proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin). Depending on the binding region, the binding agent used to measure the level of proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin) may bind to more than one of the molecules shown above. This will be clear to those skilled in the art.

Thus, according to the present invention, the level of an immunoreactive analyte obtained from a body fluid of the subject is determined by using at least one binding agent that binds to a region within the amino acid sequence of any of the above peptides and peptide fragments (i.e., proenkephalin (PENK) and fragments according to any of SEQ ID NOs: 1-12); and in association with specific clinically relevant embodiments.

In a more specific embodiment of the method according to the present invention, the level of MRPENK (SEQ ID NO. 6: proenkephalin 119-159, mid-region proenkephalin-fragment, MRPENK) is determined. In a more specific embodiment, the level of an immunoreactive analyte is determined using at least one binding agent that binds to MR-PENK, and is associated with clinically relevant embodiments according to the present invention.

Measuring the level of proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin or fragments thereof) may refer to measuring immunoreactivity against proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin). Depending on the binding region, the binding agent used to measure the level of proenkephalin or fragments thereof (including Leu-enkephalin and Met-enkephalin) may bind to more than one of the molecules shown above. This will be clear to those skilled in the art. In another embodiment of the present invention, the fragment is not Leu-enkephalin or Met-enkephalin. In another embodiment of the present invention, immunoreactivity against proenkephalin or fragments thereof (excluding Leu-enkephalin and Met-enkephalin) is measured.

In a more specific embodiment of the method according to the invention the level of MRPENK (SEQ ID NO. 6 (proenkephalin 119-159, mid-region proenkephalin-fragment, MRPENK, DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS) is determined.

Alternatively, the levels of any of the above analytes may be determined by other analytical methods such as mass spectrometry.

In a specific embodiment, the level of proenkephalin or a fragment thereof is measured using an immunoassay using an antibody or antibody fragment that binds to proenkephalin or a fragment thereof. An immunoassay that can be used to determine the level of proenkephalin or a fragment thereof having at least 5 amino acids can include the steps outlined in Example 2. All thresholds and values must be understood as being relative to the test and calibration used as described in Example 2. One skilled in the art will appreciate that the absolute value of the thresholds can be affected by the calibration used. This means that all values and thresholds given herein should be understood in the context of the calibration used herein (Example 2).

According to the present invention, the diagnostic binding agent for proenkephalin is selected from: antibodies such as IgG (a typical full-length immunoglobulin), or antibody fragments comprising at least the F-variable domains of the heavy and/or light chains, such as chemically coupled antibodies (antigen-binding fragments), including but not limited to: Fab-fragments including Fab miniantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, such as Fab-V5Sx2; bivalent Fab (miniantibodies) dimerized with a CH3 domain; bivalent Fab or multivalent Fab, for example formed by multimerization with the help of a heterologous domain, such as Fab-dHLX-FSx2 formed by dimerization of dHLX domains; F(ab')2-fragments, scFv-fragments, multimeric multivalent or/and multispecific scFv-fragments, bivalent and/or bispecific diabodies, (bispecific T cell connectors), trifunctional antibodies, multivalent antibodies, for example from types other than G; single domain antibodies such as nanobodies derived from camel or fish immunoglobulins.

In a specific embodiment, the level of proenkephalin or a fragment thereof is measured using an assay using a binding agent selected from an aptamer, a non-Ig scaffold (described in more detail below) that binds to proenkephalin or a fragment thereof.

Binding agents useful for determining the level of proenkephalin or a fragment thereof exhibit an affinity constant for proenkephalin of at least 10 ⁷ M ⁻¹ , preferably 10 ⁸ M ⁻¹ , preferably greater than 10 ⁹ M ⁻¹ , and most preferably greater than 10 ¹⁰ M ⁻¹ . Those skilled in the art will appreciate that compensating for lower affinity by administering a higher dose of the compound is contemplated and that such a measure would not exceed the scope of the present invention. Binding affinity can be determined using the Biacore method, which is available as a service assay, for example, from Biaffin, Kassel, Germany (http://www.biaffm.com/de/).

Human proenkephalin control samples can be purchased from ICI-Diagnostics, Berlin, Germany http://www.ici-diagnostics.com/ The assay can also be calibrated with synthetic (for our experiments we used synthetic MRPENK, SEQ ID NO. 6) or recombinant proenkephalin or fragments thereof.

According to the methods of the present invention, a threshold value for determining the risk of breast cancer in a female subject or diagnosing breast cancer in a female subject is less than 100 pmol/l PENK, preferably less than 50 pmol/l, and more preferably less than 40.4 pmol/l. In a specific embodiment, the threshold value is about 40.4 pmol/l. These threshold values are related to the calibration method mentioned above. A PENK value less than the threshold value means that the subject has an increased risk of cancer or has cancer.

In one embodiment of the invention, in order to monitor the risk of breast cancer in a female subject or in order to monitor the course of treatment, the method is implemented more than once. In a specific embodiment, the monitoring is carried out in order to assess the reaction of the female subject to the preventive and/or therapeutic measures taken.

In one embodiment of the invention, the method is used to stratify the female subject into risk groups.

The subject of the present invention is also a method for predicting the risk of cancer in a female or determining that a female subject has an increased risk of cancer, according to any of the preceding embodiments, wherein the level of proenkephalin or a fragment thereof having at least 5 amino acids obtained in a body fluid of said female subject is used, alone or in combination with other prognostic laboratory or clinical parameters, to predict the risk of an adverse event in the subject, by an alternative method which may be selected from the group consisting of:

compared to the median value of the level of proenkephalin or a fragment thereof having at least 5 amino acids obtained from a body fluid of said female subjects in a population of predetermined samples from a population of "healthy" or "apparently healthy" subjects,

compared to the quantile of the level of proenkephalin or a fragment thereof of at least 5 amino acids obtained from a body fluid of said female subject in a population of predetermined samples from a group of "healthy" or "apparently healthy" subjects,

• Calculations were performed based on Cox proportional hazards analysis or by using risk index calculations such as the NRI (net reclassification index) or IDI (integrated discrimination index).

In one embodiment, the present invention also provides a method for predicting the risk of cancer in a female or determining that a female subject has an increased risk of cancer according to any of the preceding embodiments, wherein the level of proenkephalin or a fragment thereof having at least 5 amino acids obtained from a body fluid of the female subject is alone or in combination with other prognostic biomarkers. Such a useful biomarker may be proneurotensin or a fragment thereof having at least 5 amino acids.

In a more specific embodiment of the method according to the invention, the level of pro-neurotensin 1-117 is determined in addition to proenkephalin and fragments thereof.

Therefore, the present invention also provides a method for predicting the risk of developing cancer in a female subject who does not have cancer or for diagnosing cancer in a female subject, comprising:

Determining the level of proenkephalin or a fragment thereof having at least 5 amino acids (including Leu-enkephalin and Met-enkephalin) in a body fluid obtained from the female subject; and

Determining the level of proneurotensin or a fragment thereof having at least 5 amino acids in a body fluid obtained from the female subject; and

correlating the levels of proenkephalin or fragments thereof and proneurotensin or fragments thereof having at least 5 amino acids with risk of developing cancer, wherein decreased proenkephalin levels predict an increased risk of developing cancer or diagnose cancer, wherein decreased levels are associated with a diagnosis of cancer, and wherein increased proneurotensin levels predict an increased risk of developing cancer or diagnose cancer, wherein increased levels are associated with a diagnosis of cancer.

SEQ ID NO.13 (Pro-neurotensin 1-147)

SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE

ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ

EDILDTGNDK NGKEEVIKRK IPYILKRQLY ENKPRRPYIL KRDSYYY

SEQ ID NO. 14 (Pro-neurotensin 1-125 (Big Neuromedin N))

SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE

ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ

EKILDTGNDK NGKEEVIKR KIPYIL

SEQ ID NO.15 (neuromediatin N)

KIPYILSEQ ID NO.16 (neurotensin)

pyroQLYENKPRRP YIL

SEQ ID NO.17 (Pro-neurotensin 1-117)

SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE

ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ

EDILDTGNDK NGKEEVI

SEQ ID NO.18 (Pro-neurotensin 1-132)

SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCS LVNNLNSPAE

ETGEVHEEEL VARRKLPTAL DGFSLEAMLT IYQLHKICHS RAFQHWELIQ

EDILDTGNDK NGKEEVIKRK IPYILKRQLY EN

SEQ ID NO.19 (pro-neurotensin 120-140)

KIPYILKRQL YENKPRRPYI L

SEQ ID NO.20 (pro-neurotensin 120-147)

KIPYILKRQL YENKPRRPYIL KRDSYYY

SEQ ID NO.21 (pro-neurotensin 128-147)

QLYENKPRRP YILKRDSYYY

In a specific embodiment, the level of proneurotensin is measured using an immunoassay. More specifically, an immunoassay is used as described in Ernst et al. (Peptides (2006), (27) 1787-1793). An immunoassay that can be used to determine the level of proneurotensin or a fragment thereof having at least 5 amino acids can include the steps outlined in Example 2. All thresholds and values must be understood as being associated with the test and calibration used as described in Example 2. As will be appreciated by those skilled in the art, the absolute values of the thresholds can be affected by the calibration used. This means that all values and thresholds given herein should be understood in the context of the calibration used herein (Example 2). Human proneurotensin-calibrator is available from ICI-Diagnostics, Berlin, Germany. Alternatively, the assay can also be calibrated using synthetic or recombinant P-NT 1-117 or a fragment thereof (see also Ernst et al, 2006).

Binding agents useful for determining the level of proneurotensin or a fragment thereof exhibit an affinity constant for proneurotensin of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M- ¹ , preferably greater than 10 ⁹ M- ¹ , and most preferably greater than 10 ¹⁰ M- ¹ . One skilled in the art will appreciate that compensating for lower affinity by administering a higher dose of the compound is contemplated and does not exceed the scope of the present invention. Binding affinity can be determined using Biacore, which is available as a service assay, for example, from Biaffin, Kassel, Germany (http://www.biaffin.com/de/). According to the methods of the present invention, a threshold value for determining the risk of breast cancer in a female subject or for diagnosing breast cancer in a female subject is greater than 78 pmol/l PNT, preferably 100 pmol/l, and more preferably 150 pmol/l. In a specific embodiment, the threshold value is approximately 100 pmol/l. These threshold values are related to the calibration methods mentioned above. A PNT value greater than the threshold value indicates that the subject has an increased risk of developing cancer or has developed cancer.

In one embodiment of the invention, in order to monitor the risk of breast cancer in a female subject, or in order to monitor a course of treatment, the method is implemented more than once. In a specific embodiment, the monitoring is carried out in order to assess the reaction of the female subject to the preventive and/or therapeutic measures taken.

In one embodiment of the invention, the method is used to stratify the female subject into risk groups.

In one embodiment of the invention, the cancer is selected from breast cancer and lung cancer.

The present invention also provides an assay for the determination of proenkephalin and proenkephalin fragments in a sample, comprising two binding agents that bind to two different regions of the proenkephalin region, namely amino acids 133-140 (LKELLETG, SEQ ID NO. 22) and amino acids 152-159 (SDNEEEVS, SEQ ID NO. 23), wherein each of said regions comprises at least 4 or 5 amino acids.

In one embodiment of the assay for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, the analytical sensitivity of the assay is capable of quantifying proenkephalin or proenkephalin fragments in healthy subjects and is <15 pmol/L, preferably <10 pmol/L, and more preferably <6 pmol/L.

In one embodiment of the assay according to the invention for determining proenkephalin or a proenkephalin fragment in a sample, the binding agent exhibits a binding affinity for its binding partner of at least 10 ⁷ M ⁻¹ , preferably 10 ⁸ M ⁻¹ , preferably with an affinity constant of less than 10 ⁹ M ⁻¹ , most preferably less than 10 ¹⁰ M ⁻¹ . A person skilled in the art will appreciate that compensating for a lower affinity by administering a higher dose of the compound may be considered, and that this measure does not lead to exceeding the scope of the present invention, and that the binding affinity may be determined as described above.

In one embodiment of the assay according to the present invention for determining proenkephalin or a proenkephalin fragment in a sample, the assay is a sandwich assay, preferably a fully automated assay. It can be an ELISA, fully automated or manual. It can be a so-called POC assay (point-of-care). Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche, Abbott, Siemens, Brahms, Biomerieux, Alere. Examples of assay formats are provided above.

In one embodiment of the assay for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, at least one of the two binding agents is labeled for detection. Examples of labels are provided above.

In one embodiment of the assay for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, at least one of the two binding agents is bound to a solid phase. Examples of solid phases are provided above.

In one embodiment of the assay for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, the label is selected from the group consisting of: a chemiluminescent label, an enzyme label, a fluorescent label, and a radioiodine label.

A further aspect of the invention is a kit comprising an assay according to the invention, wherein the components of the assay may be contained in one or more containers.

Example

Example 1

Antibody development

peptides

The peptides were synthesized (JPT Technologies, Berlin, Germany).

Peptides/conjugates for immunization:

Peptides for immunization were synthesized with an additional N-terminal cysteine amino group for conjugation to bovine serum albumin (BSA) (JPT Technologies, Berlin, Germany). Peptides were covalently linked to BSA using sulfo-SMCC (Perbio-science, Bonn, Germany). Coupling protocols were performed according to the Perbio manual.

Table 1:

Peptides for immunization Enkephalin sequence (C)DAEEDD 119-125 (C)EEDDSLANSSDLLK 121-134 (C)LKELLETG 133-140 (C)TGDNRERSHHQDGSDNE 139-155

(C)SDNEEEVS 152-159

Antibodies were generated according to the following methods:

BALB/c mice were immunized with 100 μg of peptide-BSA conjugate (emulsified in 100 μl complete Freund's adjuvant) on days 0 and 14, and 50 μg (in 100 μl incomplete Freund's adjuvant) on days 21 and 28. Three days before fusion experiments, animals received one intraperitoneal and one intravenous injection of 50 μg of the conjugate dissolved in 100 μl saline.

Spleen cells from immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded in 96-well cell culture plates. Heterozygous clones were selected by culturing in HAT medium (RPMI 1640 medium supplemented with 20% fetal calf serum and HAT supplement). After two weeks, the HAT medium was replaced with HT medium, passaged three times, and then returned to normal cell culture medium.

Three weeks after fusion, cell culture supernatants were first screened for antigen-specific IgG antibodies. Positive test microcultures were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotypes were determined.

(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 ofmonoclonal GAD antibodies", Horm. Metab. Res. 28:11-15, (1996)).

Monoclonal antibody preparation

The antibody was prepared by standard antibody production methods (Marx et al., Monoclonal Antibody Production (1997), ATLA 25, 121) and purified by protein A chromatography. The antibody purity was >95% based on SDS gel electrophoresis analysis.

Antibody labeling and coating

All antibodies were labeled with acridinium esters according to the following protocol:

Labeled compound (tracer): 100 μg (100 μl) of antibody (1 mg/ml in PBS, pH 7.4) was mixed with 10 μl of acridine NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated at room temperature for 20 min. The labeled antibody was purified by gel filtration HPLC on a Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative light units (RLU) of labeled compound (approximately 20 ng labeled antibody) per 200 μl.

Acridinium ester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Solid phase antibodies (coated antibodies):

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

Antibody Specificity:

The cross-reactivity of different antibodies is listed in Table 2.

Table 2:

Peptides for immunization preproenkephalin sequence Antibody name (C)DAEEDD 119-125 NT-MRPENK (C)EEDDSLANSSDLLK 121-134 NM-MRPENK (C)LKELLETG 133-140 MR-MRPENK (C)TGDNRERSHHQDGSDNE 139-155 MC-MRPENK (C)SDNEEEVS 152-159 CT-MRPENK

Antibody cross-reactivity was determined as follows:

1 μg of peptide in 300 μl of PBS, pH 7.4, was transferred to a polystyrene tube and incubated at room temperature for 1 hour. Following incubation, the tube was washed five times with 1 ml of 5% BSA in PBS, pH 7.4. Each labeled antibody (300 μl in PBS, pH 7.4, 800,000 RLU/300 μl) was added and incubated at room temperature for 2 hours. After five washes (each with 1 ml of wash solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X 100)), the remaining luminescence (labeled antibody) was quantified using an AutoLumat LB 953. MRPENK peptide was used as a reference substance (100%).

Table 3:

All antibodies bound to MRPENK peptides comparable to the peptides used for immunization. Apart from the NT-MRPENK-antibody (10% cross-reactivity with EEDDSLANSSDLLK), none of the antibodies showed cross-reactivity with the MR-PENK peptides not used for immunization.

Proenkephalin immunoassay:

50 μl of sample (or calibrant) was aspirated into the coated tube and, after adding labeled antibody (200 μl), the tube was incubated at 18-25°C for 2 h. Unbound tracers were removed by washing 5 times (1 ml each time) with a washing solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X-100). The labeled antibodies bound to the tube were measured using a Luminumeter LB 953 and a fixed concentration of 1000 pmol/l MRPENK. The signal (RLU, 1000 pmol MRPENK/l) and noise (RLU, no MRPENK) ratios for different antibody combinations are shown in Table 4. All antibodies were able to produce sandwich complexes with any other antibody. Surprisingly, the strongest signal-to-noise ratio (optimal sensitivity) was achieved by combining MR-MRPENK-20 and CT-MRPENK antibodies. Subsequently, we used this antibody combination to perform MRPENK-immunoassay for further study. MR-MRPENK antibody was used as the tube coating antibody, and CT-MRPENK antibody was used as the labeled antibody.

Table 4:

calibration:

The assay was calibrated using a solution of synthetic MRPENK _diluted in 20 mM _K2PO4 , 6 mM EDTA, 0.5% BSA, 50 μΜ aminopeptidase inhibitor, 100 μΜ leupeptin, pH 8.0. Proenkephalin control plasma was purchased from ICI-diagnostics, Berlin, Germany.

A typical proenkephalin dose/signal curve is shown in Figure 1. Standard curve proenkephalin.

The assay sensitivity was 5.5 pmol/L (0 minus calibrator (without MRPENK added) + 2SD) after 20 measurements.

Group studies

method

Methods: Fasting plasma proenkephalin was measured in 2559 female participants (age 58 ± 6 years, 59% female) from the population-based Malmo Diet and Cancer Study at baseline between 1991 and 1994. We used multivariable-adjusted (all conventional cardiovascular risk factors, diabetes risk factors, and analyses of cancer and heritability) Cox proportional hazards models to associate baseline PENK (hazard ratio per standard deviation increase in log-transformed PENK) with the time to the first event of each study endpoint during a median follow-up of more than 12 years. Endpoints were obtained by searching the Swedish National Hospital Discharge Registry, the Swedish Myocardial Infarction Registry, the Stroke in Malmo Registry, and the Swedish Cancer Registry. Endpoints obtained through these registry searches were validated and found to be accurate (see also Belting et al. Cancer Epidemiol Biomarkers Prev; 1-10. 2012 AACR).

Clinical characteristics of the women in the study

Table 5:

Figure 2: Frequency distribution of proenkephalin in the female population:

The mean value was 47.2 pmol/L with a standard deviation of 1.2 pmol/L. The x-axis is the natural logarithm (LN) of the PENK concentration. All results were within the range of the assay, with the lowest PENK concentration being 9 pmol/L. These results indicate the suitability of the assay used (assay sensitivity was 5.5 pmol/L).

PENK and breast cancer prediction

We evaluated the relationship between proenkephalin and breast cancer (Table 6). There was a strong association between proenkephalin and breast cancer in women. In the fully adjusted model, each SD increase in proenkephalin was associated with a 28.6% reduced risk, or each SD decrease in proenkephalin (revPENK) was associated with a 40% increased risk of future breast cancer (Table 5), and the highest to lowest quartile ratio of proenkephalin determined a more than 3-fold difference in breast cancer risk (see Table 7 and Figure 3).

Table 6:

The variable ^D in the equation

Table 7:

Multivariate Cox proportional hazards model of baseline proenkephalin relative to breast cancer incidence.

Figure 3: Kaplan Meier graph showing cumulative breast cancer diagnoses for women in quartile 1 (Q) (less than 40.4 pmol/l), quartile 2 (40.4-47.1 pmol/l), quartile 3 (47.2-54.1 pmol/l), and quartile 4 (greater than 54.1 pmol/l). Reduced PENK indicates a long-term increased risk of breast cancer development. Because any women with a history of cancer on the day of baseline (blood sampling) were excluded, proenkephalin was highly predictive of future breast cancer development. In summary, women from Q1 had a 3.6-fold higher risk of developing breast cancer compared to women from Q4.

Combined proenkephalin and proneurotensin

Since increased proneurotensin was recently shown to be highly predictive for breast cancer, we combined biomarkers for breast cancer prediction.

Example

Proneurotensin assay

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

Immunoassay for the Quantification of Human Proneurotensin

The technology used was an acridinium ester-labeled sandwich-coated tube luminescence immunoassay.

Labeled compound (tracer): 100 μg (100 μl) of LA (1 mg/ml in PBS, pH 7.4) was mixed with 10 μl of acridine NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP0353971) and incubated at room temperature for 20 min. Labeled LA was purified by gel filtration HPLC on a Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA). Purified LA was diluted in (300 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative light units (RLU) of labeled compound (approximately 20 ng of labeled antibody) per 200 μl. Acridinium ester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

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

calibration:

The assay was calibrated using dilutions of proneurotensin containing human serum.A human serum pool with high proneurotensin immunoreactivity (InVent Diagostika, Hennigsdorf, Germany) (assay standard) was diluted with horse serum (Biochrom AG, Germany).

The standard was calibrated by using human pro-neurotensin-calibrator (ICI-Diagnostics, Berlin, Germany). Alternatively, the assay can be calibrated by synthetic or recombinant P-NT 1-117 or fragments thereof (see also Ernst et al., 2006).

ProNT immunoassay:

50 μl of sample (or calibrant) was pipetted into a SPA-coated tube. After adding labeled LA (200 μl), the tube was incubated at 18-25°C for 16-22 hours. Unbound tracer was removed by washing five times (1 ml each) with wash solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X-100). LA bound to the tube was measured using a Luminumeter LB 953. Results were calculated from the calibration curve.

Combined analysis of proenkephalin and PNT in female population:

There was no significant correlation between proenkephalin and proneurotensin (p=0.56).In a combined model, using both biomarkers, we found that they were independent in breast cancer prediction.

In the fully adjusted model, each SD increase in PNT was associated with a 49.9% increased risk of future breast cancer. Surprisingly, after adding PNT to the equation, PENK was even stronger than without PNT, showing that each SD increase in proenkephalin was associated with a 30.8% decreased risk, or each SD decrease in proenkephalin (revPENK) was associated with a 44.5% increased risk of future breast cancer (Table 8).

Table 8: Combined analysis of PNT and PENK for breast cancer prediction.

Table 8: Variables in the equation

The highest versus lowest quartile of PNT indicated a 2.56-fold risk of breast cancer development, and proenkephalin in the upper lowest versus highest quartile of PNT (rev = reversed quartile Q1 = Q4, Q2 = Q3, Q3 = Q2, Q4 = Q1) was independently a 3.6-fold risk (Table 9).

The highest quartile of combined PNT and the lowest proenkephalin quartile showed a combined risk of 6.17 relative to the lowest PNT- and highest proenkephalin quartiles (see Figure 3).

Table 9: Combined analysis of PNT and PENK for breast cancer prediction.

Table 9:

Variables in the equation

Figure 4: Example of combined analysis of proenkephalin for breast cancer prediction:

We combined women with the lowest (1st) proenkephalin quartile and the highest (4th) proneurotensin quartile (Group 3). In the high-risk group, approximately 19.02% of women developed breast cancer over the subsequent 15 years.

Group 2 was a combination of women with proneurotensin in quartile 3 and proenkephalin in quartile 2 plus proneurotensin in quartile 2 and proenkephalin in quartile 3. In the intermediate-risk group, approximately 7.48% of women developed breast cancer over the subsequent 15 years.

Group 1 was a combination of women with pro-neurotensin levels in the first quartile and pro-enkephalin levels in the fourth quartile. In this low-risk group, approximately 3.08% of women developed breast cancer over the subsequent 15 years. The hazard ratio between the first and third groups was approximately 6.17.

lung cancer

Proenkephalin also predicts lung cancer in women.

40 women developed lung cancer during the observation period. There was no difference in proenkephalin between smokers and nonsmokers (p = 0.44). As expected, smoking was a strong risk predictor for lung cancer (p < 0.0001). Surprisingly, despite smoking being part of the equation, low proenkephalin indicated a 3.2-fold increased risk of developing lung cancer (Tables 10a and 10b).

Tables 10a and 10b: PENK prediction of lung cancer in women. Women were divided into 3 groups (see Table 10a) and then analyzed for lung cancer development (see Table 10b). rev = highest tertile (3rd tertile), rev(1) = 2nd tertile, and rev(2) = lowest tertile (1st tertile).

Table 10a:

PENK [pmol/L]

Table 10b:

Variables in the equation

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Shows a typical proenkephalin dose/signal curve. Standard curve proenkephalin.

Figure 2: Frequency distribution of proenkephalin in the female population:

Figure 3: Kaplan Meier graph showing cumulative breast cancer diagnoses for women in quartile 1 (Q) (less than 40.4 pmol/l), quartile 2 (40.4-47.1 pmol/l), quartile 3 (47.2-54.1 pmol/l), and quartile 4 (greater than 54.1 pmol/l). Reduced PENK indicates a long-term increased risk of breast cancer development. Because any women with a history of cancer on the day of baseline (blood sampling) were excluded, proenkephalin was highly predictive of future breast cancer development. In summary, women from Q1 had a 3.6-fold higher risk of developing breast cancer compared to women from Q4.

Figure 4: Example of combined analysis of proenkephalins for breast cancer prediction.

Claims (22)

1. Use of a binder in the preparation of a reagent for predicting the risk of cancer in a female subject who has not yet developed cancer, wherein the binder is capable of measuring the level of proenkephalin or at least five amino acid fragments thereof obtained from bodily fluids of the female subject, and wherein the proenkephalin or at least five amino acid fragments thereof are selected from the group comprising the sequences: 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 and SEQ ID No. 11, wherein the cancer is breast cancer or lung cancer. 2. The use as claimed in claim 1, wherein the female subject had never had a history of cancer diagnosis when the bodily fluid sample was collected from the female subject. 3. The use as claimed in claim 1, wherein the female subject has a history of cancer diagnosis and has been cured at the time the bodily fluid sample was collected from the female subject, and the risk of recurrence of breast cancer has been determined. 4. The use as described in any one of claims 1-3, wherein the female subject has been diagnosed with cardiovascular disease or diabetes at the time of collecting the bodily fluid sample from the female subject. 5. The use as described in any one of claims 1-3, wherein at least one clinical parameter selected from the group consisting of: age, presence of diabetes, and current smoking is further defined. 6. The use as claimed in claim 4, wherein at least one clinical parameter selected from the group consisting of: age, presence of diabetes, and current smoking is further identified. 7. The use as described in any one of claims 1-3, wherein the level of proenkephalins is measured using an immunoassay. 8. The use as described in any one of claims 1-3, wherein the reagent is used more than once for monitoring the risk of breast cancer in a female subject or for monitoring the treatment process. 9. The use as claimed in claim 8, wherein the monitoring is performed to assess the female subject's response to preventive and/or therapeutic measures. 10. The use as described in any one of claims 1-3, for classifying the female subject into a risk group. 11. The use according to any one of claims 1-3, wherein the reagent further comprises a binder for determining the level of pro-nervone 1-117 (SEQ ID No. 17) obtained from the bodily fluids of the female subject. 12. The use as described in any one of claims 1-3, wherein a reduced level of proenkephalin or a fragment thereof predicts an increased risk of cancer, which is a level below a threshold, wherein the threshold is less than 100 pmol/L. 13. The use as claimed in claim 11, wherein the increased level of neurotensinogen or a fragment thereof predicts an increased risk of cancer, which is a level above a threshold, wherein the threshold is greater than 78 pmol/L. 14. The use as claimed in claim 12, wherein the increased level of neurotensinogen or a fragment thereof predicts an increased risk of cancer, which is a level above a threshold, wherein the threshold is greater than 78 pmol/L. 15. The use as described in any one of claims 1-3, wherein the body fluid is blood, plasma, or serum. 16. The use as described in any one of claims 1-3, wherein the levels of MR-enkephalin (SEQ ID No. 6) and/or neurotensinogen 1-117 (SEQ ID No. 17) were determined. 17. The use as claimed in claim 11, wherein the levels of MR-enkephalin (SEQ ID No. 6) and/or neurotensinogen 1-117 (SEQ ID No. 17) were determined. 18. A kit for determining proenkephalin and proenkephalin fragments in a sample, comprising two binding agents that bind to two different regions of the proenkephalin region, namely amino acids 133-140 (LKELLETG, SEQ ID NO. 22) and amino acids 152-159 (SDNEEEVS, SEQ ID NO. 23). 19. The kit of claim 18, wherein the kit is sufficiently sensitive to quantify proenkephalin or proenkephalin fragments in healthy subjects and has an analytical sensitivity of <10 pmol/L. 20. The kit of claim 18 or 19, wherein the levels of the enkephalinogen and enkephalinogen fragments are used to predict the risk of cancer in a female subject who has not had cancer, and wherein the cancer is breast cancer or lung cancer. 21. Use of two binding agents in the preparation of a reagent for predicting the risk of cancer in female subjects without cancer, and/or for classifying said female subjects into risk groups, wherein said two binding agents bind to two different regions of the proenkephalin region, namely amino acids 133-140 (LKELLETG, SEQ ID NO. 22) and amino acids 152-159 (SDNEEEVS, SEQ ID NO. 23). 22. Use of the two binding agents in the preparation of a reagent for classifying female subjects into groups that should receive opioid growth factor (OGF) treatment, wherein the two binding agents bind to two distinct regions of the enkephalin origin region, namely amino acids 133-140 (LKELLETG, SEQ ID NO. 22) and amino acids 152-159 (SDNEEEVS, SEQ ID NO. 23).