Method for predicting the risk of getting cancer or diagnosing cancer in a subject
10605812 ยท 2020-03-31
Assignee
Inventors
Cpc classification
G01N33/74
PHYSICS
G01N2410/00
PHYSICS
International classification
G01N31/00
PHYSICS
G01N33/53
PHYSICS
G01N33/74
PHYSICS
Abstract
A method for predicting the risk of getting cancer in a subject that does not suffer from cancer or alternatively diagnosing cancer in a subject, where the method includes determining the level of Pro-Tachykinin, its splice variants or fragments thereof of at least 5 amino acids, where the fragments including Substance P and Neurokinin, in a bodily fluid obtained from the subject; and correlating the level of Pro-Tachykinin, its splice variants or fragments thereof with a risk for getting cancer, wherein a reduced Pro-Tachykinin level is predictive for an enhanced risk of getting cancer or alternatively diagnosing cancer wherein a reduced level is correlated with the diagnosis of cancer.
Claims
1. A method for predicting the risk of getting cancer in a subject that does not suffer from cancer comprising: determining the level of Pro-Tachykinin in a bodily fluid sample obtained from said subject by determining the level of binding to the region of Pro-Tachykinin that consists of Pro-Tachykinin 1-37 (SEQ ID NO. 2) by measuring the level with an immunoassay having an antibody specific to binding to SEQ ID NO. 2; and correlating said level of Pro-Tachykinin with risk for getting cancer, wherein a reduced level is predictive for an enhanced risk of getting cancer and said reduced level is a level below a threshold wherein said threshold is set at 100 pmol/l or below.
2. The method according to claim 1 further comprising the following steps: determining the level of Pro-Neurotensin in a bodily fluid obtained from said subject; and correlating additionally Pro-Neurotensin with a risk for getting cancer, wherein an increased level of 78 pmol/l or above of Pro-Neurotensin is predictive for an enhanced risk of getting cancer.
3. The method according to claim 1 further comprising the following steps: determining the level of Pro-Enkephalin in a bodily fluid obtained from said subject; and correlating additionally Pro-Enkephalin with a risk for getting cancer, and wherein a reduced level of 100 pmol/l or below of Pro-Enkephalin is predictive for an enhanced risk of getting cancer.
4. The method according to claim 1 further comprising the following steps: determining the level of Insulin in a bodily fluid obtained from said subject; and correlating additionally said level of Insulin with a risk for getting cancer, wherein a reduced level of 70 pmol/l or below of Insulin is predictive for an enhanced risk of getting cancer.
5. The method according to claim 2 wherein additionally correlating means a combined analysis of the determined biomarker levels by taking into account the relative risk factors for cancer development obtained by the individual biomarkers.
6. The method according to claim 1 wherein a reduced level of Pro-Tachykinin is a level below a threshold wherein said threshold is set at 80 pmol/L or below.
7. The method according to claim 2 wherein an increased level of Pro-Neurotensin is a level above a threshold wherein said threshold is 100 pmol/l.
8. The method according to claim 3 wherein a reduced level of Pro-Enkephalin is a level below a threshold wherein said threshold is 75 pmol/L.
9. The method according to claim 1 wherein said subject is female.
10. The method according to claim 9, wherein said cancer is breast cancer.
11. The method according to claim 1 wherein said cancer is lung cancer.
12. A The method according to claim 1, wherein said subject has never had a history of diagnosis of cancer at the time the sample of bodily fluid is taken from said subject.
13. The method according to claim 1, wherein said subject has had a history of diagnosis of cancer and has been cured at the time the sample of bodily fluid is taken from said subject and the risk of reoccurrence of getting cancer is determined or alternatively the reoccurrence of breast cancer is determined.
14. The method according to claim 1, wherein at the time the sample of bodily fluid is taken from said subject, said subject has been diagnosed as having a cardiovascular disease or diabetes.
15. The method according to claim 1, wherein additionally at least one clinical parameter is determined selected from: age, presence of diabetes mellitus, and current smoking.
16. The method according to claim 2, wherein the level of Pro-Neurotensin is measured with an immunoassay.
17. The method according to claim 1 wherein said method is performed more than once on the same subject later in time in order to monitor the risk of getting cancer in the subject or in order to monitor the course of treatment.
18. The method according to claim 17 wherein said monitoring is performed in order to evaluate the response of said subject to preventive and/or therapeutic measures taken.
19. The method according to claim 1, wherein multiple reduced level thresholds of Pro-Tachykinin are determined in order to stratify said subjects into multiple differing risk groups.
20. The method according to claim 1 wherein the bodily fluid is blood or plasma or serum.
21. The method according to claim 1 wherein a reduced level of Pro-Tachykinin is a level below a threshold wherein said threshold is set at 60 pmol/L or below.
22. The method according to claim 1 wherein a reduced level of Pro-Tachykinin is a level below a threshold wherein said threshold is set at 50 pmol/L or below.
23. The method according to claim 2 wherein an increased level of Pro-Neurotensin is a level above a threshold wherein said threshold is 150 pmol/l.
24. The method according to claim 3 wherein a reduced level of Pro-Enkephalin is a level below a threshold wherein said threshold is 50 pmol/L.
25. The method according to claim 3, wherein the level of Pro-Enkephalin is measured with an immunoassay.
26. The A method according to claim 4, wherein the level of Insulin is measured with an immunoassay.
27. The method according to claim 1, wherein the subject has a reduced level of Pro-Tachykinin below 100 pmol/l and the subject is given preventative or therapeutic treatment for cancer.
Description
FIGURE DESCRIPTION
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EXAMPLES
Example 1 PTA-immunoassay
(6) Development of Anti PTA Antibodies
(7) Peptides/Conjugates for Immunization:
(8) Peptides for immunization were synthesized OPT 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.
(9) TABLE-US-00007 TABLE1 Peptideforimmunization PTASequence (C)GANDDLNYWSDWYDSDQIK 3-22(SEQIDNO.12) (C)IKEELPEPFEHLLQRI 21-36(SEQIDNO.13)
(10) The antibodies were generated according to the following method:
(11) A BALB/c mouse was immunized with 100 g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 l complete Freund's adjuvant) and 50 g at day 21 and 28 (in 100 l incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 g of the conjugate dissolved in 100 l saline, given as one intraperitonal and one intravenous injection.
(12) Splenocytes 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.
(13) 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.
(14) (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)).
(15) Monoclonal Antibody Production
(16) 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.
(17) Labelling and Coating of Antibodies.
(18) All antibodies were labelled with acridinium ester according the following procedure:
(19) Labelled compound (tracer, anti PTA 3-22): 100 g (100 l) antibody (1 mg/ml in PBS, pH 7.4, was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
(20) Solid Phase Antibody (Coated Antibody):
(21) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with anti PTA 22-36 antibody (1.5 g antibody/0.3 ml 100 mmol/l NaCl, 50 mmol/l Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.
(22) PTA Immunoassay:
(23) 50 l of sample (or calibrator) was pipetted into coated tubes, after adding labeled antibody (200ul), 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/1 PBS, pH 7.4, 0.1% TRITON X-100 nonionic surfactant). Tube-bound labelled antibody was measured by using a Luminumeter LB 953, Berthold, Germany.
(24) Calibration:
(25) The assay was calibrated, using dilutions of synthetic P37, diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 M Amastatin, 100 M Leupeptin, pH 8.0. PTA control plasma is available at ICI-diagnostics, Berlin, Germany.
(26)
(27) The analytical assay sensitivity was (the median signal generated by 20 determinations of 0-calibrator (no addition of PTA)+2SD2 standard deviations (SD), the corresponding PTA concentration is calculated from a standard curve) 4.4 pmol/L.
Example 2 Population Study/PTA
(28) Methods
(29) We measured PTA in fasting plasma from 2559 female participants of the population based Malm Diet and Cancer Study baseline exam in 1991-1994 (age 586 years). 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 PTA (hazard ratio per each standard deviation increase of log-transformed PTA) to the time to the first event of each of the studied endpoints during a median follow-up time of more than 12 years. Endpoints were retrieved through the Swedish National Hospital Discharge Registry, the Swedish Myocardial Infarction Registry, the Stroke in Malm Registry and the Swedish Cancer Registry. Retrieval of endpoints through these registries has been validated and found to be accurate (see also Belting et al. Cancer Epidemiol Biomarkers Prev; 1-10. 2012 AACR). Insulin was measured by standard laboratory methods.
(30) TABLE-US-00008 TABLE 2 Clinical characteristics of females in the study: Descriptive Statistics N Mean Std. Deviation Age at MDCS screening 2559 57.554 5.9403 Systolic blood pressure (mmHg) 2559 140.50 19.311 Diastolic blood pressure (mmHg) 2559 85.65 9.117 body-mass-index (weight/kg kg) 2559 25.5196 4.19083 WAIST (cm) 2559 76.99 10.245 Glucose (mmol/l) 2559 5.0418 1.21798 Triglycerides (mmol/l) 2559 1.2245 .58404 High density lipoprotein (mmol/l) 2559 1.5123 .36949 Low density lipoprotein (mmol/l) 2559 4.2016 1.04762 P-Insulin 2512 7.223 5.4223
Distribution of PTA in the Females Population (N=2559):
(31) The mean value of PTA in the female population was 54.3 pmol/L, standard deviation+/1.4 pmol/L. All results were within the measurement range of the assay, the lowest PTA concentration was 9.1 pmol/L. These results indicating the suitability of the used assay (assay sensitivity 4.4 pmol/L).
(32) PTA and Prediction of Breast Cancer
(33) We assessed the relationship between PTA and breast cancer (Table 3). All women with previous cancer (N=459) were excluded from the evaluation. There was a strong relationship between PTA and breast cancer in females. In a fully adjusted model each SD of decrease of PTA (we used reversed quartiles, revPTA, see table 3/4) was associated with a 28.2% increased risk of future breast cancer (table 3) and the top versus bottom quartile of PTA identified a more than 2.1-fold difference in risk of breast cancer (see table 5 and
(34) TABLE-US-00009 TABLE 3 Variables in the Equation 95.0% CI B SE Wald df Sig. Exp (B) Lower AGE .003 .016 .035 1 .851 .997 .966 BMI_B .027 .025 1.194 1 .275 1.027 .979 LNINS .423 .200 4.465 1 .035 .655 .442 HER_CANCER_0 .006 .184 .001 1 .973 .994 .693 Q_REV_PTA .249 .085 8.629 1 .003 1.282 1.086
(35) TABLE-US-00010 TABLE 4 PTA Quartile analysis: Concentration range Quartile Rev Quartile N (pmol PTA/l) 1 4 535 <45.6 2 3 535 45.6-55.3 3 2 535 55.4-65.9 4 1 535 >65.9
(36) TABLE-US-00011 TABLE 5 Multivariate Cox proportional Hazards models for baseline PTA versus incidence of breast cancer. HR per 1 SD P-value Quartile 4 Quartile 3 Quartile 2 Quartile 1 Women 1.22 0.013 1.0 (ref) 1.60 1.6 2.2 (2140/137) (0.84-1.67) (1.21-2.22) (1.24-2.27) (1.82-3.6)
Example 3
(37) Pro-Neurotensin Assay
(38) Antibodies were generated as described above. The antibody for labelling (LA) was generated against P-NT 1-19 (H-CSDSEEEMKALEADFLTNMH (SEQ ID NO. 33)) and the solid phase antibody (SPA) was generated against peptide P-NT 44-62 (CNLNSPAEETGEVHEEELVA (SEQ ID NO. 34). Antibody development and -production was performed as described above.
(39) Immunoassay for the Quantification of Human Pro-Neurotensin
(40) The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.
(41) Labelled compound (tracer): 100 g (100 l) LA (1 mg/ml in PBS, pH 7.4, was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled LA was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified LA was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
(42) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with SPA (1.5 g SPA/0.3 ml 100 mmol/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.
(43) Calibration:
(44) 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).
(45) The standards were calibrated by use of the human Pro-Neurotensin-calibrator (ICI-Diagnostics, Berlin, Germany). Alternatively, the assay may be calibrated by synthetic or recombinant P-NT 1-117 or fragments thereof (see also Ernst et al., 2006).
(46) ProNT Immunoassay:
(47) 50 l of sample (or calibrator) was pipetted into SPA coated tubes, after adding labelled LA (200ul), 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 nonionic surfactant). Tube-bound LA was measured by using a Luminometer LB 953. Results were calculated from the calibration curve. A typical calibration curve is shown in
Example 4
(48) Pro-Enkephalin Immunoassay
(49) Development of Antibodies
(50) Peptides/Conjugates for Immunization:
(51) Peptides for immunization were synthesized OPT 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.
(52) TABLE-US-00012 TABLE6 Pro-Enkephalin- Peptideforimmunization sequence (C)LKELLETG(SEQIDNO.35) 133-140 (C)SDNEEEVS(SEQIDNO.36) 152-159
(53) The antibodies were generated according to the following method:
(54) A BALB/c mouse was immunized with 100 g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 l complete Freund's adjuvant) and 50 g at day 21 and 28 (in 100 l incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 g of the conjugate dissolved in 100 l saline, given as one intraperitonal and one intravenous injection.
(55) Splenocytes 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.
(56) 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.
(57) (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)).
(58) TABLE-US-00013 TABLE7 Pre-Pro- Peptidefor Enkephalin- immunization sequence Antibodyname (C)LKELLETG 133-140 MR-MRPENK (SEQIDNO.35) (usedascoated tubeantibody) (C)SDNEEEVS 152-159 CT-MRPENK (SEQIDNO.36) (usedaslabelled antibody)
Monoclonal Antibody Production
(59) 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.
(60) Labelling and Coating of Antibodies.
(61) Labelled compound (tracer, CT-MRPENK antibody): 100 g (100 l) antibody (1 mg/ml in PBS, pH 7.4), was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
(62) Solid Phase Antibody (Coated Tube Antibody, MR-MRPENK Antibody):
(63) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 g antibody/0.3 ml 100 mmol/l NaCl, 50 mmol/l Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.
(64) Pro-Enkephalin Immunoassay:
(65) 50 l of sample (or calibrator) was pipetted into coated tubes, after adding labelled antibody (200ul), 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 nonionic surfactant). Tube-bound labelled antibody was measured by using the Luminometer 953.
(66) Calibration:
(67) The assay was calibrated, using dilutions of synthetic MRPENK, diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 M Amastatin, 100 M Leupeptin, pH 8.0. Pro-Enkephalin control plasma is available at ICI-diagnostics, Berlin, Germany.
(68)
(69) The assay sensitivity (20 determinations of 0-calibrator (no addition of MRPENK)+2SD) was 5.5 pmol/L.
Example 5
(70) Combination Analysis of PTA, Pro Neurotensin and HRT and, PTA, Pro-Neurotensin, Pro-Enkephalin and Insulin for Breast Cancer Prediction
(71) Since increasing Pro-Neurotensin and Pro-Enkephalin recently were shown to be highly predictive for breast cancer, we combined these biomarkers for breast cancer prediction. We added HRT (Hormone replacement therapy) as known risk factor for breast cancer to show the incremental value of PTA.
(72) First, we combined PTA/ProNeurotensin/HRT/Insulin:
(73) There was no significant correlation between PTA and Pro-Neurotensin (p=0.71). In a combined model including Insulin and hormone replacement therapy (HRT) using PTA and PNT (Table 8), we found them both independent in breast cancer prediction. Both markers were highly significant (p=0.005 for PTA and p<0.001 for PNT).
(74) In a fully adjusted model each SD increase of PNT was associated with a 45.5% risk increase of future breast cancer. Each SD increase of PTA was associated with a 18.9% decreased risk (per SD) of future breast cancer.
(75) HRT, as expected, was significant in the same model, but Insulin, surprisingly, was on top predicting breast cancer (p=0.027). Each SD increase of Insulin was associated with a 35.7% decrease of future breast cancer.
(76) These data show that PTA, PNT, Insulin and HRT, each add significant information for breast cancer prediction.
(77) TABLE-US-00014 TABLE 8 combined analysis of PNT and PTA for breast cancer prediction. Variables in the Equation 95.0% Exp CI B SE Wald df Sig. (B) Lower AGE .004 .016 .048 1 .826 .996 .966 BMI_B .029 .025 1.342 1 .247 1.029 .980 LNINS .441 .199 4.889 1 .027 .643 .435 hrt_curr .612 .197 9.656 1 .002 1.844 1.254 HER_CANCER_0 .014 .184 .006 1 .938 1.014 .707 ZLN_PTA .210 .075 7.925 1 .005 .811 .700 ZLN_PNT .375 .089 17.938 1 .000 1.455 1.223
(78) In a Kaplan Meier analysis we illustrate the combinatory information of PTA and PNT, Table 9 and
(79) We combined quartiles of PTA and PNT:
(80) Since low PTA values indicating an increased risk of breast cancer development, we reversed the PTA quartiles (revPTA): 1.sup.st quartile PTA=4.sup.th quartile revPTA; 2.sup.nd quartile PTA=3.sup.rd quartile revPTA; 3.sup.rd quartile PTA=2.sup.nd quartile revPTA; 4.sup.th quartile PTA=1.sup.st quartile revPTA. (Table 9)
(81) TABLE-US-00015 TABLE 9 Relative risk N of 15 year breast risk (lowest risk revPTA/PNT subjects cancer development (%) group = 1) Q1/Q1 117 3 2.6 1 (group 1) Q1/Q2 and 673 27 4.0 1.54 Q2/Q1 Q1/Q3 Q2/Q2 Q3/Q1 (group 2) Q1/Q4 583 42 7.2 2.8 Q2/Q3 Q3/Q2 Q4/Q1 (group 3) Q2/Q4 377 25 6.6 2.5 Q3/Q3 Q4/Q2 (group 4) Q3/Q4 263 24 9.1 3.5 Q4/Q3 (group 5) Q4/Q4 127 16 12.6 4.9 (group 6)
(82) Combining highest quartile of PNT and lowest PTA quartile (group 6) vs. lowest PNT- and highest PTA quartile (group 1) showed a combined risk of 4.9 for future breast cancer (see
(83) Combined Analysis of PTA, Pro Enkephalin, HRT, Insulin and PNT in the Female Population:
(84) There was a significant correlation between PTA and Pro-Enkephalin (p=<0.001, r=0.35). In a combined model including Insulin, PTA, PNT and Pro-Enkephalin, we found all markers independently adding information for breast cancer prediction (Table 10). All markers were highly significant (p=0.028 for PTA, p<0.001 for PNT, p=0.009 for Insulin and p<0.001 for Pro Enkephalin). PTA remains independent although it is highly correlated to Pro Enkephalin. In a fully adjusted model each SD increase of PNT was associated with a 47.8% risk increase of future breast cancer. Increase of PTA was associated with a 15.8% decreased risk (per SD) of future breast cancer. Increase of Pro-Enkephalin was associated with a decreased risk of 26.4% (per SD)- and increase of Insulin was associated with a decreased risk of 40.4% (per SD) of future breast cancer.
(85) These data show a strong independent and additive information on future breast cancer development by PTA, PNT, Pro-Enkephalin and Insulin.
(86) TABLE-US-00016 TABLE 10 combined analysis of PTA, Pro-Enkephalin, Insulin and PNT. Variables in the Equation 95.0% Exp CI B SE Wald df Sig. (B) Lower AGE .001 .016 .002 1 .966 .999 .969 BMI_B .019 .025 .568 1 .451 1.019 .970 LNINS .518 .200 6.739 1 .009 .596 .403 HER_CANCER_0 .021 .185 .012 1 .911 .980 .682 ZLN_PTA .171 .078 4.827 1 .028 .842 .723 ZLN_PNT .390 .089 19.328 1 .000 1.478 1.242 ZLN_PENK .309 .087 12.655 1 .000 .734 .619