DIAGNOSTIC METHOD

Abstract

The disclosure is directed to a method of diagnosing a prostate condition in a subject by determining, in a sample obtained from a subject, levels of a plurality of constituents selected from the group consisting of Ca, K, Mg, Zn, Ag, AI, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, and Zr. A combination of the levels of the plurality of constituents in the sample is compared with a combination of control levels of the same plurality of constituents. A difference between the combinations is indicative of the prostate condition.

Claims

1. A method of diagnosing a prostate condition in a subject, comprising: determining, in a sample obtained from a subject, levels of a plurality of constituents selected from the group consisting of Ca, K, Mg, Zn, Ag, Al, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, and Zr; and comparing a combination of the levels of the plurality of constituents in the sample with a combination of control levels of the same plurality of constituents, in which a difference between the combinations is indicative of the prostate condition.

2.-18. (canceled)

19. A method according to claim 1, wherein the prostate condition is prostate cancer.

20. A method according to claim 19, wherein the sample obtained from the subject comprises seminal fluid or expressed prostatic secretion.

21. A method according to claim 19, comprising determining levels of least five of the constituents.

22. A method according to claim 19, comprising determining levels of at least six of the constituents.

23. A method of diagnosing a prostate condition in a subject, comprising: determining, in sample obtained from a subject, a level of at least one constituent selected from the group consisting of Ca, K, Mg, Ag, Al, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y and Zr; and comparing the level of the at least one constituent in the sample with a control level of the same at least one constituent, in which a difference between the level of the at least one constituent in the sample and the control level of the same at least one constituent is indicative of the prostate condition, wherein the sample comprises seminal fluid or expressed prostatic secretion.

24. A method according to claim 23, wherein the prostate condition is prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The appended drawings have been included herein so that the cited features, advantages, and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

[0064] FIG. 1 shows individual data sets for Al mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0065] FIG. 2 shows individual data sets for Ba mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0066] FIG. 3 shows individual data sets for Bi mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0067] FIG. 4 shows individual data sets for Ca mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0068] FIG. 5 shows individual data sets for Mg mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0069] FIG. 6 shows individual data sets for Mn mass fractions (mg/kg of dry tissue) in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0070] FIG. 7 shows individual data sets for Ca/Fe mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0071] FIG. 8 shows individual data sets for Mg/AI mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0072] FIG. 9 shows individual data sets for Ca/Cu mass fraction in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0073] FIG. 10 shows individual data sets for (Ca/Cu)*(Mg/Cu) mass fraction ratios combination in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0074] FIG. 11 shows individual data sets for (Ca/Cu)*(Zn/Cu) mass fraction ratios combination in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0075] FIG. 12 shows individual data sets for (Mg/Cu)*(Zn/Cu) mass fraction ratios combination in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0076] FIG. 13 shows individual data sets for Ca/Ba mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0077] FIG. 14 shows individual data sets for Ca/P mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0078] FIG. 15 shows individual data sets for Ca/Si mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0079] FIG. 16 shows individual data sets for Ca/Sr mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0080] FIG. 17 shows individual data sets for Zn/Mn mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0081] FIG. 18 shows individual data sets for [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0082] FIG. 19 shows individual data sets for [(Ca*Cd*Co*Hg*K*Mg*Na*P*Rb*S*Se*Zn)/(Ag*Al*Ba*Bi*Br*Ce*Cr*Cs*Cu*Li*Mn*Ni*Pb*Sb*S i*Sr)]*10.sup.18 in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0083] FIG. 20 shows individual data sets for [(Ca*Cd*Co*Hg*K*Mg*Na*P*Rb*S*Sc*Se*Zn)/(Ag*Al*Au*B*Ba*Bi*Br*Ce*Cr*Cs*Cu*Dy*Er*F e*Gd*Ho*La*Li*Mn*Nd*Ni*Pb*Pr*Sb*Si*Sm*Sr*Th*TI*U*Y*Zr)]*10.sup.34 in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

[0084] FIG. 21 shows individual data sets for normalized mass fraction additive indices of four selected elements in samples of normal, benign hyperplastic and cancerous EPS.

[0085] FIG. 22 shows individual data sets for normalized mass fraction multiplicative indices of four selected elements in samples of EPS from normal, benign hyperplastic and cancerous patients.

[0086] FIG. 23 shows individual data sets for normalized mass fraction multiplicative indices of six selected elements in samples of EPS from normal, benign hyperplastic and cancerous patients.

[0087] FIG. 24. Individual data sets for product index (Rb*Zn)/10 obtained from the EPS samples from healthy (1), benign hypertrophic (2) and cancerous individuals (3).

[0088] The following examples are given for the purpose of illustrating the various embodiments of the present invention and are not meant to limit the present invention in any fashion.

SPECIFIC EXAMPLES

Example 1

Identification of Cancer Biomarkers in Prostate Tissue Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Atomic Emission Spectrometry (ICP-AES)

[0089] Experimental conditions of the present study were approximated to the hospital conditions as closely as possible.

[0090] Equipment:

[0091] Autoclave (Ancon-AT2, Russia), inductively coupled plasma mass spectrometry instrument Thermo-Fisher X-7 (Thermo Electron, USA), Spectrometer ICAP-61 (Thermo Jarrell Ash, USA).

[0092] Specimen:

[0093] Benign prostate hyperplasia samples (n=43) and prostate adenocarcinoma samples (n=60) were obtained by transrectal biopsy of an indurated site of prostate. Samples of the human normal prostate tissue (n=37) were obtained at autopsy of male patients aged 41-87 died of an injury or in a car accident. The presence or absence of cancer in tissue samples was confirmed by microscopic analysis of tissue morphology.

[0094] Reagents:

[0095] HNO.sub.3 (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H.sub.2O.sub.2 (hydrogen peroxide pure for analysis, Merck), ICP-MS standards ICP-MS-68A and ICP-AM-6-A (High-Purity Standards, Charleston, S.C. 29423, USA), ICP stock solutions (High-Purity Standards, Charleston, S.C. 29423, USA).

[0096] Protocol:

[0097] 1.5 mL of HNO.sub.3 and 0.3 mL of H.sub.2O.sub.2 were added to homogenized and freeze-dried prostate tissue sample, placed in one-chamber autoclave, and decomposed for 3 hours at 160-200 C. The heat-treated sample was cooled down to the room temperature; the soluble fraction was diluted with deionized water to 20 mL and transferred to a plastic measuring bottle. Simultaneously, the same procedure was performed on a sample containing no prostate tissue, and the resultant solution was used as a blank sample. All samples were analysed by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

[0098] The spectrometer parameters and the main parameters of ICP-MS measurements: generator output power 1,250 W, spray chamber cooled at 3 C., plasma gas flow rate12 L/min, nebuliserPolycon, auxiliary air flow rate0.9 L/min, nebulizer flow rate0.9 L/min, sample update0.8 mL/min, resolution0.8, detector modedouble, scanning modesurvey scan (number of runs10, dwell time0.6 ms, channels per mass10, acquisition duration13.2 s) and peak jumping (sweeps25, dwell time10 ms, channels per mass1, acquisition duration34 s).

[0099] The spectrometer parameters for ICP-AES measurements: generator output power 1,200 W, reflected power<5 W, nebuliser typeangular, plasma-forming air flow rate18 L/min, auxiliary air flow rate0.9 L/min, air flow rate into atomiser0.6 L/min, flow rate of the analysed sample1.5 mL/min, zone height for plasma observation14 mm.

[0100] Results:

[0101] The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, Sb, Sc, Se, Si, Sm, Th, Tl, U, Y, and Zr in prostate tissue was analysed by ICP-MS. The content of Na, Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in prostate tissue was analysed by ICP-AES.

[0102] Statistically significant differences in mass fraction levels of 45 chemical elements (Table 1) were found in samples derived from cancerous, benign hyperplastic and normal prostate tissues. Differences in mass fraction levels of these elements can be used for diagnosis and therapeutic purpose. The data in Table 1 allow evaluating the importance of the individual chemical element content information for the diagnosis of PCa.

TABLE-US-00001 TABLE 1 Comparison of mean values (M SEM) of chemical element mass fractions (mg .Math. kg.sup.1, dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) prostate tissue Prostate tissue Ratios, p (t-test) Normal BPH PCa BPH PCa PCa 41-87 year 38-83 year 40-79 year to to to Element n = 37 n = 43 n = 60 Normal Normal BPH Ag 0.0284 0.0035 0.0407 0.0088 0.255 0.031 1.43 8.98.sup.c 6.27.sup.c Al 34.1 3.5 24.4 3.2 328 73 0.72 9.62.sup.c 13.4.sup.c Au 0.0041 0.0008 0.0026 0.0008 0.0297 0.0056 0.63 7.24.sup.c 11.4.sup.c B 1.04 0.18 1.51 0.26 12.6 3.7 1.45 12.1.sup.b 8.34.sup.b Ba 1.48 0.21 1.22 0.20 26.7 7.6 0.82 18.0.sup.b 21.9.sup.b Bi 0.029 0.011 0.140 0.042 1.76 0.27 4.83.sup.a 60.7.sup.c 16.9.sup.c Br 27.9 2.9 30.0 2.6 99.9 8.9 1.08 3.58.sup.c 3.33.sup.c Ca 2397 235 2032 165 675 58 0.85 0.28.sup.c 0.33.sup.c Cd 1.12 0.12 1.07 0.43 0.425 0.099 0.96 0.38.sup.c 0.40 Ce 0.0309 0.0050 0.0128 0.0019 0.101 0.013 0.41.sup.b 3.27.sup.c 7.89.sup.c Co 0.0452 0.0043 0.0716 0.0097 0.0326 0.0037 1.58.sup.a 0.72.sup.a 0.46.sup.b Cr 0.53 0.08 1.07 0.12 2.35 0.37 2.02.sup.c 4.43.sup.c 2.20.sup.b Cs 0.0339 0.0033 0.0235 0.0025 0.0389 0.0039 0.69.sup.a 1.14 1.66.sup.b Cu 9.85 0.97 9.86 1.25 17.1 2.0 1.00 1.74.sup.b 1.74.sup.b Dy 0.0029 0.0005 0.0016 0.0002 0.0072 0.0011 0.53.sup.a 2.48.sup.c 4.50.sup.c Er 0.00148 0.00023 0.00072 0.00013 0.00297 0.00038 0.49.sup.b 2.01.sup.b 4.13.sup.c Fe 111 9 139 10 165 15 1.25 1.49.sup.a 1.19 Gd 0.0029 0004 0.0015 0.0003 0.0094 0.0017 0.52.sup.b 3.24.sup.c 6.27.sup.c Hg 0.052 0.009 0.275 0.036 0.130 0.021 5.29.sup.c 2.50.sup.c 0.47.sup.b Ho 0.00057 0.00008 0.00032 0.00005 0.00178 0.00022 0.56.sup.a 3.12.sup.c 5.56.sup.c K 12030 475 14472 740 8542 504 1.20.sup.b 0.71.sup.c 0.59.sup.c La 0.080 0.019 0.019 0.003 0.970 0.540 0.24.sup.b 12.1 51.1 Li 0.0419 0.0055 0.0385 0.0073 0.251 0.054 0.92 5.99.sup.b 6.52.sup.a Mg 1071 76 1201 83 346 61 1.12 0.32.sup.c 0.29.sup.c Mn 1.32 0.08 1.19 0.09 6.99 1.35 0.90 5.30.sup.c 5.87.sup.c Na 10987 394 11612 869 7511 643 1.06 0.68.sup.c 0.65.sup.c Nd 0.0137 0.0021 0.0062 0.0009 0.0413 0.0065 0.45.sup.b 3.01.sup.c 6.66.sup.c Ni 3.10 0.51 3.22 1.06 6.96 1.04 1.04 2.25.sup.c 2.16.sup.b P 7617 368 7907 418 6675 465 1.04 0.88 0.84 Pb 2.39 0.56 0.69 0.16 1.81 0.35 0.29.sup.a 0.76 2.62.sup.b Pr 0.0035 0.0005 0.0015 0.0003 0.0097 0.0017 0.43.sup.b 2.77.sup.b 6.47.sup.c Rb 14.8 0.9 14.4 0.7 8.8 0.7 0.97 0.59.sup.c 0.61.sup.c S 8557 254 8787 487 5343 389 1.03 0.62.sup.c 0.61.sup.c Sb 0.037 0.005 0.142 0.036 0.501 0.062 3.84.sup.b 13.5.sup.c 3.53.sup.c Sc 0.0294 0.0053 0.0257 0.0040 0.0116 0.0015 0.87 0.39.sup.b 0.45.sup.b Se 0.696 0.044 1.243 0.079 0.576 0.078 1.79.sup.c 0.83 0.46.sup.c Si 102 11 141 24 284 39 1.38 2.78.sup.c 2.02.sup.b Sm 0.0027 0.0004 0.0014 0.0004 0.0095 0.0029 0.52.sup.a 3.52.sup.a 6.71.sup.b Sr 2.34 0.38 3.69 0.45 5.75 0.60 1.58.sup.a 2.46.sup.c 1.56.sup.a Th 0.0034 0.0007 0.0018 0.0003 0.0490 0.0120 0.52.sup.a 14.4.sup.c 27.2.sup.c Tl 0.0014 0.0002 0.0020 0.0006 0.0219 0.0056 1.43 15.6.sup.c 11.0.sup.b U 0.0070 0.0021 0.0021 0.0009 0.0068 0.0013 0.30.sup.a 0.97 3.24.sup.b Y 0.0186 0.0042 0.0071 0.0012 0.0340 0.0038 0.38.sup.a 1.83.sup.b 4.79.sup.c Zn 1061 153 1136 96 136 9.9 1.07 0.13.sup.c 0.12.sup.c Zr 0.036 0.006 0.091 0.036 2.13 0.89 2.53 59.2.sup.a 23.4.sup.a M-arithmetic mean, SEM-standard error of mean, .sup.ap 0.05, .sup.bp 0.01, .sup.cp 0.001.

Example 2

Establishing the Prostate Condition using Al Mass Fraction in Prostate Tissue Sample

[0103] The tissue content of Al was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Al in tissue of normal prostate was found to be 34.13.5 (SEM) mg/kg, in BPH 24.43.2 (SEM) mg/kg, and in PCa 32873 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Al mass fraction in dry normal and BPH prostate tissue was determined to be M+2SD or 70 mg/kg on dry mass basis (FIG. 1).

[0104] If PCa needs to be discriminated from normal and BPH tissue and if Al content in a prostate biopsy sample prepared and analyzed as described in the Example 1 exceeds 70 mg/kg dry tissue, prostate carcinoma with an accuracy of 8212% can be diagnosed. The sensitivity and specificity of the Al based test is 973% and 944%, respectively.

Example 3

Establishing the Prostate Condition Using Ba Mass Fraction in Prostate Tissue Sample

[0105] The tissue content of Ba was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Ba in tissue of normal prostate was found to be 1.480.21 (SEM) mg/kg, in BPH 1.220.20 (SEM) mg/kg, and in PCa 26.77.6 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Ba mass fraction in dry normal and BPH prostate tissue was determined to be M+2SD or 3.5 mg/kg on dry mass basis (FIG. 2).

[0106] If PCa needs to be discriminated from normal and BPH tissue and if Ba content in a prostate biopsy sample prepared and analyzed as described in the Example 1 exceeds 3.5 mg/kg dry tissue, prostate carcinoma with an accuracy of 8212% can be diagnosed. The sensitivity and specificity of the Ba based test is 973% and 944%, respectively.

Example 4

Establishing the Prostate Condition Using Bi Mass Fraction in Prostate Tissue Sample

[0107] The tissue content of Bi was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Bi in tissue of normal prostate was found to be 0.0290.011 (SEM) mg/kg, in BPH 0.1400.042 (SEM) mg/kg, and in PCa 1.760.27 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Bi mass fraction in dry normal and BPH prostate tissue was determined to be M+2SD or 0.5 mg/kg on dry mass basis (FIG. 3).

[0108] If PCa needs to be discriminated from normal and BPH tissue and if Bi content in a prostate biopsy sample prepared and analyzed as described in the Example 1 exceeds 0.5 mg/kg dry tissue, prostate carcinoma with an accuracy of 8212% can be diagnosed. The sensitivity and specificity of the Bi based test is 973% and 934%, respectively.

Example 5

Establishing the Prostate Condition Using Ca Mass Fraction in Prostate Tissue Sample

[0109] The tissue content of Ca was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Ca in tissue of normal prostate was found to be 2397235 (SEM) mg/kg, in BPH 2032165 (SEM) mg/kg, and in PCa 67558 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Ca mass fraction in dry cancerous prostate tissue was determined to be M+2SD or 1080 mg/kg on dry mass basis (FIG. 4).

[0110] If PCa needs to be discriminated from normal and BPH tissue and if Ca content in a prostate biopsy sample prepared and analysed as described in the Example 1 does not exceed 1080 mg/kg dry tissue, prostate carcinoma with an accuracy of 98% can be diagnosed. The sensitivity and specificity of the Ca based test is 98% and 97%, respectively.

Example 6

Establishing the Prostate Condition Using Mg Mass Fraction in Prostate Tissue Sample

[0111] The tissue content of Mg was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Mg in tissue of normal prostate was found to be 10717 (SEM) mg/kg, in BPH 120183 (SEM) mg/kg, and in PCa 34661 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Mg mass fraction in dry cancerous prostate tissue was determined to be M+2SD or 700 mg/kg on dry mass basis (FIG. 5).

[0112] If PCa needs to be discriminated from normal and BPH tissue and if Mg content in a prostate biopsy sample prepared and analysed as described in the Example 1 does not exceed 700 mg/kg dry tissue, prostate carcinoma with an accuracy of 904% can be diagnosed. The sensitivity and specificity of the Mg based test is 100-10% and 846%, respectively.

Example 7

Establishing the Prostate Condition Using Mn Mass Fraction in Prostate Tissue Sample

[0113] The tissue content of Mn was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1, Table 1). Mass fraction of Mn in tissue of normal prostate was found to be 1.320.08 (SEM) mg/kg, in BPH 1.190.09 (SEM) mg/kg, and in PCa 6.991.35 (SEM) mg/kg on dry mass basis (Table 1). The upper limit for Mn mass fraction in dry normal or BPH prostate tissue was determined to be M+2SD or 2 mg/kg on dry mass basis (FIG. 6).

[0114] If PCa needs to be discriminated from normal and BPH tissue and if Mn content in a prostate biopsy sample prepared and analysed as described in the Example 1 exceeds 2 mg/kg dry tissue, prostate carcinoma with an accuracy of 963% can be diagnosed. The sensitivity and specificity of the Mn based test is 919% and 973%, respectively.

Example 8

Determination of Mass Fraction Levels of 44 Elements Relative to the Mass Fraction of Calcium in Normal, Cancerous and BPH Prostate Tissue

[0115] Mass fraction ratios of the elements mentioned in the Example 1 are different in non-cancerous and cancerous tissue and therefore these can be used as prostate tumor biomarkers. In the Table 2 mass fraction ratios of 44 elements relative to mass fraction of calcium are presented. Further, ratios of the mass fraction ratios for normal prostate tissue, BPH and cancerous tissue are given. The mass fraction ratios presented in the Table 2 is a mean of ratios calculated for every single prostate sample. The data in the Table 2 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of calcium for the diagnosis of PCa.

TABLE-US-00002 TABLE 2 Means of ratios (M SEM), their ratios and the reliability of difference between mean values of mass fraction ratios of Ca to mass fractions of other chemical element in normal, benign hyperplastic (BPH) and cancerous (PCa) prostate tissue. Prostate tissue Ratios of means, p (-test) Mass Normal BPH PCa BPH PCa PCa fraction 41-79 year 38-83 year 40-79 year to to to ratio n = 37 n = 43 n = 60 Normal Normal BPH Ca/Ag 107037 15763 117550 34515 4164 1611 1.10 0.039.sup.c 0.035.sup.b Ca/Al 103 21 101 18 5.24 1.9 0.98 0.051.sup.c 0.052.sup.c Ca/B 4320 805 1550 191 119 58 0.36.sup.b 0.028.sup.c 0.077.sup.c Ca/Ba 2957 577 2034 251 102 47 0.69 0.035.sup.c 0.050.sup.c Ca/Bi 532698 114578 93934 41193 7501 6139 0.18.sup.c 0.014.sup.c 0.080.sup.a Ca/Br 91.3 12.7 78.9 14.6 11.1 4.8 0.86 0.12.sup.c 0.14.sup.c Ca/Cd 3085 455 3753 732 2002 212 1.22 0.65.sup.a 0.53.sup.a Ca/Ce 144087 28909 191735 31186 8862 2222 1.33 0.062.sup.c 0.046.sup.c Ca/Co 69329 11034 42314 4982 13669 1072 0.61.sup.a 0.20.sup.c 0.32.sup.c Ca/Cr 14516 6572 2169 218 191 41 0.15 0.013.sup.a 0.088.sup.c Ca/Cs 94882 17335 92843 9485 22723 6474 0.98 0.24.sup.c 0.24.sup.c Ca/Cu 315 47 223 23 56 14 0.71 0.18.sup.c 0.25.sup.c Ca/Dy 1733952 444593 1685620 327920 161389 47689 0.97 0.093.sup.c 0.096.sup.c Ca/Er 2782727 557202 3989832 845199 296667 64924 1.43 0.11.sup.c 0.074.sup.c Ca/Fe 20.8 2.4 17.8 1.9 4.81 0.53 0.86 0.23.sup.c 0.27.sup.c Ca/Gd 1489869 334486 1726552 327682 118454 35677 1.16 0.080.sup.c 0.069.sup.c Ca/Hg 78186 11882 9944 1259 4445 2330 0.13.sup.c 0.057.sup.c 0.45.sup.a Ca/Ho 7482530 1547065 8358623 1644445 453653 88284 1.12 0.061.sup.c 0.054.sup.c Ca/K 0.227 0.037 0.144 0.013 0.081 0.008 0.63.sup.a 0.36.sup.c 0.56.sup.c Ca/La 105705 19452 128328 16885 7340 3488 1.21 0.069.sup.c 0.057.sup.c Ca/Li 79700 10834 71782 11904 5847 2025 0.90 0.073.sup.c 0.082.sup.c Ca/Mg 2.83 0.51 1.72 0.12 2.58 0.47 0.61 0.91 1.50 Ca/Mn 2061 325 1789 186 181 66 0.87 0.088.sup.c 0.10.sup.c Ca/Na 0.236 0.032 0.189 0.025 0.097 0.014 0.80 0.41.sup.c 0.51.sup.b Ca/Ni 1916 626 1028 179 123 28 0.54 0.064.sup.b 0.12.sup.c Ca/P 0.348 0.040 0.264 0.025 0.112 0.020 0.76 0.32.sup.c 0.42.sup.c Ca/Pb 3774 724 4461 756 556 149 1.18 0.15.sup.c 0.12.sup.c Ca/Pr 1263853 269288 2231480 735010 112525 35218 1.77 0.089.sup.c 0.050.sup.b Ca/Rb 180 23 139 12 81.8 7.8 0.77 0.45.sup.c 0.59.sup.c Ca/S 0.308 0.045 0.238 0.022 0.133 0.016 0.77 0.43.sup.c 0.56.sup.c Ca/Sb 121963 24741 49028 20319 2784 556 0.40.sup.a 0.023.sup.c 0.057.sup.a Ca/Sc 174958 51707 76930 10995 49945 6858 0.44 0.29.sup.a 0.65.sup.a Ca/Se 3604 500 2362 296 895 168 0.66.sup.a 0.25.sup.c 0.38.sup.c Ca/Si 35.7 6.9 17.4 2.3 3.21 0.88 0.49.sup.a 0.090.sup.c 0.18.sup.c Ca/Sm 1695658 438262 2939100 810027 142073 37596 1.73 0.084.sup.b 0.048.sup.c Ca/Sr 1334 142 743 76 137 34 0.56.sup.b 0.10.sup.c 0.18.sup.c Ca/Th 1703628 375869 1499284 275745 43707 21300 0.88 0.026.sup.c 0.029.sup.c Ca/Tl 2870569 627543 1464103 252751 124174 74903 0.51.sup.a 0.043.sup.c 0.085.sup.c Ca/U 1122953 182815 2061625 434930 130793 22073 1.84 0.12.sup.c 0.063.sup.c Ca/Y 414438 116105 385834 74931 23034 3863 0.93 0.056.sup.b 0.060.sup.c Ca/Zn 3.89 0.91 1.72 0.21 5.02 0.41 0.44.sup.a 1.29 2.92.sup.c Ca/Zr 135701 31300 61766 18949 853 238 0.46.sup.a 0.0063.sup.c 0.014.sup.c M-arithmetic mean, SEM-standard error of mean, .sup.ap 0.05, .sup.bp 0.01, .sup.cp 0.001.

Example 9

Determination of Mass Fraction Levels of 44 Elements Relative to Mass Fraction of Zinc in Normal, Cancerous and BPH Prostate Tissue

[0116] Mass fraction ratios of the elements mentioned in the Example 1 are different in non-cancerous and cancerous tissue and therefore these can be used as prostate tumor biomarkers. In the Table 3 mass fraction ratios of 44 elements relative to mass fraction of zinc are presented. Further, ratios of the mass fraction ratios for normal prostate tissue, BPH and cancerous tissue are given. The mass fraction ratios presented in the Table 3 is a mean of ratios calculated for every single prostate sample. The data in the Table 3 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of Zn for the diagnosis of PCa.

TABLE-US-00003 TABLE 3 Means of ratios (M SEM), their ratios and the reliability of difference between mean values of mass fraction ratios of Zn to mass fractions of other chemical element in normal, benign hyperplastic (BPH) and cancerous (PCa) prostate tissue Prostate tissue Ratios, p (t-test) Mass Normal BPH PCa BPH PCa PCa fraction 41-79 year 38-83 year 40-79 year to to to ratio n = 37 n = 43 n = 60 Normal Normal BPH Zn/Ag 32271 5360 39748 4328 723 133 1.23 0.022.sup.c 0.018.sup.c Zn/Al 41.3 9.7 59.0 9.8 1.16 0.52 1.43 0.028.sup.c 0.020.sup.c Zn/Au 645790 240530 816590 173610 19120 12210 1.27 0.029.sup.b 0.023.sup.c Zn/B 1974 559 1360 417 28.8 21.0 0.69 0.015.sup.b 0.021.sup.b Zn/Ba 1003 195 1373 203 30 17 1.37 0.030.sup.c 0.022.sup.c Zn/Bi 236160 62300 79960 37890 2290 2110 0.34.sup.a 0.0097.sup.c 0.029.sup.a Zn/Br 39.1 6.2 68.8 11.5 1.30 0.14 1.76.sup.a 0.033.sup.c 0.019.sup.c Zn/Ca 0.449 0.059 0.758 0.171 0.169 0.027 1.69 0.38.sup.c 0.22.sup.b Zn/Cd 39170 11900 39100 6460 319 59 1.00 0.0082.sup.c 0.0082.sup.c Zn/Ce 60330 14060 131210 22300 2055 899 2.17.sup.b 0.035.sup.c 0.016.sup.c Zn/Co 27011 3716 20798 3359 4293 554 0.77 0.16.sup.c 0.21.sup.c Zn/Cr 2654 356 1161 156 78.1 13.4 0.44.sup.c 0.029.sup.c 0.067.sup.c Zn/Cs 37990 8990 69050 15160 3899 1158 1.82 0.103.sup.c 0.057.sup.c Zn/Cu 114 19 133 19 9.0 2.3 1.17 0.079.sup.c 0.068.sup.c Zn/Dy 657590 148330 1194200 255540 30310 10770 1.81 0.046.sup.c 0.025.sup.c Zn/Er 1190700 293660 2796660 590750 54040 15720 2.35.sup.a 0.045.sup.c 0.019.sup.c Zn/Fe 8.8 1.4 11.8 1.5 0.97 0.11 1.34 0.11.sup.c 0.082.sup.c Zn/Gd 624740 158040 1190620 240260 23210 8250 1.91 0.037.sup.c 0.019.sup.c Zn/Hg 27011 3717 6490 688 1216 115 0.24.sup.c 0.045.sup.c 0.19.sup.c Zn/Ho 3128380 766220 5591120 958640 97340 37950 1.79 0.031.sup.c 0.017.sup.c Zn/K 0.086 0.016 0.109 0.024 0.0135 0.0026 1.27 0.16.sup.c 0.12.sup.c Zn/La 61550 25120 96666 23120 2156 1250 1.57 0.035.sup.a 0.022.sup.c Zn/Li 35526 9597 51562 11566 1248 528 1.45 0.035.sup.b 0.024.sup.c Zn/Mg 1.01 0.14 1.33 0.35 0.38 0.50 1.32 0.38 0.29 Zn/Mn 847 210 1261 185 43 23 1.49 0.051.sup.c 0.034.sup.c Zn/Na 0.099 0.019 0.144 0.035 0.0153 0.0025 1.45 0.15.sup.c 0.11.sup.b Zn/Nd 133860 32890 275460 44660 4690 1810 2.05.sup.a 0.035.sup.c 0.017.sup.c Zn/Ni 712 185 820 220 26 11 1.15 0.037.sup.c 0.032.sup.c Zn/P 0.128 0.016 0.198 0.041 0.0187 0.0042 1.55 0.15.sup.c 0.094.sup.c Zn/Pb 1523 348 2910 470 128 58 1.91.sup.a 0.084.sup.c 0.044.sup.c Zn/Pr 529125 130900 1429530 348740 21630 7700 2.70.sup.a 0.041.sup.c 0.015.sup.c Zn/Rb 71.7 9.0 87.4 9.3 17.9 2.0 1.22 0.26.sup.c 0.22.sup.c Zn/S 0.123 0.025 0.182 0.041 0.0213 0.0035 1.48 0.17.sup.c 0.12.sup.c Zn/Sb 34333 6156 10115 2344 334 44 0.29.sup.c 0.0097.sup.c 0.033.sup.c Zn/Sc 46794 7866 39678 3372 13157 1624 0.85 0.28.sup.c 0.33.sup.c Zn/Se 1548 166 886 90 270 28 0.57.sup.b 0.18.sup.c 0.30.sup.c Zn/Si 15.8 4.9 14.3 4.6 0.69 0.33 0.91 0.044.sup.c 0.048.sup.b Zn/Sm 642630 151110 1796120 413200 27835 9730 2.79.sup.a 0.043.sup.c 0.015.sup.c Zn/Sr 561 83 641 223 22.2 4.7 1.14 0.040.sup.c 0.035.sup.b Zn/Th 721180 196050 1041050 219340 12010 7530 1.44 0.017.sup.c 0.012.sup.c Zn/Tl 855140 116010 877340 132760 35010 26220 1.03 0.041.sup.c 0.040.sup.c Zn/U 461270 98980 1514290 378280 23375 6170 3.28.sup.b 0.050.sup.c 0.015.sup.c Zn/Y 171540 52620 271240 54800 4540 1495 1.58 0.026.sup.b 0.017.sup.c Zn/Zr 49100 9570 41930 11960 151 58 0.85 0.0031.sup.c 0.0036.sup.c M-arithmetic mean, SEM-standard error of mean, .sup.ap 0.05, .sup.bp 0.01, .sup.cp 0.001.

Example 10

Using the Ca/Fe Mass Fraction Ratio to Establish Prostate Condition

[0117] The Ca/Fe mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Fe mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 10 (FIG. 7).

[0118] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/Fe ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 10, prostate carcinoma with an accuracy of 963% can be diagnosed. The sensitivity and specificity of the Ca/Fe ratio based test is 100-9% and 954%, respectively.

Example 11

Using the Mg/Al Mass Fraction Ratio to Establish Prostate Condition

[0119] The Mg/Al mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for Mg/Al mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+2SD (Marithmetic mean, SDstandard deviation) or 9 (FIG. 8).

[0120] If PCa needs to be discriminated from normal and BPH tissue and if the Mg/Al ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 9, prostate carcinoma with an accuracy of 99% can be diagnosed. The sensitivity and specificity of the Mg/Al ratio based test is 98% and 99%, respectively.

Example 12

Using the Ca/Cu Mass Fraction Ratio to Establish Prostate Condition

[0121] The Ca/Cu mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Cu mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 100 (FIG. 9).

[0122] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/Cu ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 100, prostate carcinoma with an accuracy of 973% can be diagnosed. The sensitivity and specificity of the Ca/Cu ratio based test is 919% and 100-3%, respectively.

Example 13

Using the (Ca/Cu)*(Mg/Cu) Mass Fraction Ratio Combination to Establish Prostate Condition

[0123] The (Ca/Cu)*(Mg/Cu) mass fraction ratio combination was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for (Ca/Cu)*(Mg/Cu) mass fraction ratio combination on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 4000 (FIG. 10).

[0124] If PCa needs to be discriminated from normal and BPH tissue and if the (Ca/Cu)*(Mg/Cu) ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 4000, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the (Ca/Cu)*(Mg/Cu) ratio based test is 100-11% and 100-3%, respectively.

Example 14

Using the (Ca/Cu)*(Zn/Cu) Mass Fraction Ratio Combination to Establish Prostate Condition

[0125] The (Ca/Cu)*(Zn/Cu) mass fraction ratio combination was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for (Ca/Cu)*(Zn/Cu) mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 1700 (FIG. 11).

[0126] If PCa needs to be discriminated from normal and BPH tissue and if the (Ca/Cu)*(Zn/Cu) ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 1700, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the (Ca/Cu)*(Zn/Cu) ratio based test is 100-10% and 100-3%, respectively.

Example 15

Using the (Mg/Cu)*(Zn/Cu) Mass Fraction Ratio Combination to Establish Prostate Condition

[0127] The (Mg/Cu)*(Zn/Cu) mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for (Mg/Cu)*(Zn/Cu) mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 975 (FIG. 12).

[0128] If PCa needs to be discriminated from normal and BPH tissue and if the (Mg/Cu)*(Zn/Cu) ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 975, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the (Mg/Cu)*(Zn/Cu) ratio based test is 100-11% and 100-3%, respectively.

Example 16

Using Bodily Fluids and Tissues to Establish Prostate Condition

[0129] Using the method of analysis described in the Example 1 the mass fraction ratios of Ca and Mg were determined in main histological compartments of the prostate tissue: glandular epithelium, stroma and lumen. The correlation coefficients (r-value) between the mass fraction of the element in a prostate tissue compartment and the relative volume of the main histological compartments of prostate tissue are given in the Table 4. For Ca and Mg a strong correlation with lumen was found indicating that the content of given markers is reflected in the prostatic fluid, which is the main part of the content of the prostate tissue lumen. Prostatic fluid is the part of the ejaculate and is present in urine too; therefore the concentration of the specific biomarkers will also be reflected in ejaculate and urine. As a result, prostate condition can be established using the biomarkers given in the Table 1 using prostatic fluid, seminal fluid and urine samples.

TABLE-US-00004 TABLE 4 Correlation coefficient (r-value) between the mass fraction of Ca and Mg in prostate tissue and the relative volume of the main histological compartments of prostate tissue. Compartment/Element Ca Mg Glandular epithelium 0.194 0.385 Stroma -0.421 -0.482 Lumen 0.582 0.437 Statistically significant r-values are given in bold

Example 17

Identification of Cancer Biomarkes in Expressed Prostatic Secretion Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Atomic Emission Spectrometry (ICP-AES)

[0130] Experimental conditions of the present study were approximated to the hospital conditions as closely as possible.

[0131] Equipment:

[0132] Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

[0133] Specimen:

[0134] Expressed Prostatic Secretion samples (EPS) from patients with Benign Prostate Hyperplasia (BPH) and prostate adenocarcinoma (PCa) and EPS samples from healthy volunteers were obtained by transrectal prostate massage. The presence of cancer was confirmed by Digital Rectal Examination (DRE), TransRectal Ultrasound Imaging (TRUSI) and microscopic analysis of tissue morphology in biopsies obtained from the same patients. The absence of cancer was confirmed by DRE and TRUSI.

[0135] Reagents:

[0136] HNO.sub.3 (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H.sub.2O.sub.2 (hydrogen peroxide pure for analysis, Merck), ICP-MS standards NCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureau of Reference of the European Comission) and IRM-BD151 (LGC Standards, Weisel, Germany).

[0137] Protocol:

[0138] 0.5 mL of HNO.sub.3 was added to freeze-dried EPS samples and the samples were left over night at room temperature. After that 0.25 mL of HNO.sub.3 and 0.15 mL of H.sub.2O.sub.2 were added to the samples and placed in water bath at 95 C. for 30 min. The heat-treated samples were cooled down to the room temperature; the soluble fraction was diluted with deionized water to 15 mL and transferred to a plastic measuring bottle. Simultaneously, the same procedure was performed on a sample containing no EPS fluid, and the resultant solution was used as a blank sample. All samples were analysed by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

[0139] The spectrometer parameters and the main parameters of ICP-MS measurements: auxiliary air flow rate0.9 L/min, nebulizer flow rate0.9 L/min, sample update0.8 mL/min. The spectrometer parameters for ICP-AES measurements: generator output power 1,500 W.

[0140] Results:

[0141] The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Tb, Th, Tl, U, Y, and Zr in EPS was analysed by ICP-MS. The content of Na, Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in EPS was analysed by ICP-AES.

[0142] Statistically significant differences in mass fraction levels of 46 chemical elements (Table 5) were found in samples derived from cancerous, benign hyperplastic and normal prostate EPS. Differences in mass fraction levels of these elements can be used for diagnosis and therapeutic purpose. The data in Table 5 allow evaluating the importance of the individual chemical element content information for the diagnosis of prostate cancer (PCa).

TABLE-US-00005 TABLE 5 Comparison of mean values of chemical element mass fractions (mg .Math. kg.sup.1, dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) EPS. Ratios of means Prostatic fluid BPH to PCa to PCa to Element Normal BPH PCa Normal Normal BPH Li 0.45 0.18 0.21 0.4 0.5 1.2 B 0.97 2.54 1.00 2.6 1.0 0.4 Na 45440 47804 167000 1.1 3.7 3.5 Mg 5130 4549 1900 0.9 0.4 0.4 Al 4.63 12.50 43.85 2.7 9.5 3.5 Si 25.80 44.33 110.00 1.7 4.3 2.5 P 1732 4844 1800 2.8 1.0 0.4 S 5469 5063 6300 0.9 1.2 1.2 K 37920 27525 19000 0.7 0.5 0.7 Ca 11040 10758 3800 1.0 0.3 0.4 Sc 0.06 0.05 0.01 1.0 0.2 0.2 Cr 0.61 0.90 23.74 1.5 39.1 26.4 Mn 1.10 0.71 3.95 0.7 3.9 5.6 Fe 15.7 26.4 370.0 1.7 23.6 14.0 Co 0.03 0.05 0.08 1.3 2.5 1.9 Ni 0.32 4.58 7.03 14.2 21.9 1.5 Cu 8.46 13.65 14.45 1.6 1.7 1.1 Zn 8606 5099 2000 0.6 0.2 0.4 Se 1.56 1.45 0.10 0.9 0.1 0.1 Br 31.8 43.5 559.6 1.4 17.6 12.9 Rb 52.8 32.3 23.9 0.6 0.5 0.7 Sr 2.71 2.57 3.15 0.9 1.2 1.2 Y 0.01 0.01 0.03 1.3 2.6 2.0 Zr 0.07 0.14 0.47 2.1 6.8 3.3 Ag 0.03 0.37 0.94 14.4 36.2 2.5 Sb 0.10 0.51 0.10 5.1 1.0 0.2 Cs 0.08 0.08 0.10 1.1 1.3 1.2 Ba 0.35 0.35 3.31 1.0 9.4 9.4 La 0.06 0.02 0.04 0.4 0.8 2.3 Ce 0.01 0.02 0.08 1.2 6.2 5.1 Pr 0.06 0.01 0.02 0.2 0.4 2.0 Nd 0.01 0.03 0.04 2.5 3.7 1.5 Sm 0.01 0.01 0.02 1.1 2.4 2.3 Gd 0.01 0.01 0.04 1.2 3.6 3.0 Tb 0.01 0.01 0.01 1.0 1.4 1.4 Dy 0.01 0.01 0.02 1.0 1.9 1.9 Ho 0.01 0.01 0.01 1.0 1.2 1.2 Er 0.01 0.01 0.03 1.0 2.5 2.5 Au 0.07 0.19 0.01 2.6 0.1 0.1 Cd 0.03 0.08 0.37 3.3 14.4 4.3 Hg 0.08 0.05 0.0001 0.7 0.001 0.001 Tl 0.01 0.01 0.15 0.9 13.8 15.3 Pb 0.08 0.32 1.03 3.7 12.2 3.3 Bi 0.02 0.01 0.40 0.6 24.5 39.5 Th 0.03 0.01 0.04 0.5 1.7 3.8 U 0.01 0.01 0.05 1.0 4.8 4.8

Example 18

Identification of Cancer Biomarkers in Seminal Fluid Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Atomic Emission Spectrometry (ICP-AES)

[0143] Experimental conditions of the present study were approximated to the hospital conditions as closely as possible.

[0144] Equipment:

[0145] Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

[0146] Specimen:

[0147] Ejaculate samples from patients with Benign Prostatic Hyperplasia, prostate adenocarcinoma and from healthy volunteers were obtained by masturbation into a clean metal-free vial. The presence of cancer was confirmed by DRE, TRUSI and microscopic analysis of tissue morphology in biopsies obtained from the same patients. The absence of cancer was confirmed by DRE and TRUSI.

[0148] Reagents:

[0149] HNO.sub.3 (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H.sub.2O.sub.2 (hydrogen peroxide pure for analysis, Merck), ICP-MS standards NCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureau of Reference of the European Comission) and IRM-BD151 (LGC Standards, Weisel, Germany).

[0150] Protocol:

[0151] 0.5 mL of HNO.sub.3 was added to freeze-dried seminal fluid samples and the samples were left over night at room temperature. After that 0.25 mL of HNO.sub.3 and 0.15 mL of H.sub.2O.sub.2 were added to the samples and placed in water bath at 95 C. for 30 min. The heat-treated sample was cooled down to the room temperature; the soluble fraction was diluted with deionized water to 15 mL and transferred to a plastic measuring bottle. Simultaneously, the same procedure was performed on a sample containing no seminal fluid, and the resultant solution was used as a blank sample. All samples were analysed by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

[0152] The spectrometer parameters and the main parameters of ICP-MS measurements: auxiliary air flow rate0.9 L/min, nebulizer flow rate0.9 L/min, sample update0.8 mL/min. The spectrometer parameters for ICP-AES measurements: generator output power 1,500 W.

[0153] Results:

[0154] The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Tb, Th, Tl, U, Y, and Zr in seminal fluid was analysed by ICP-MS. The content of Na, Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in seminal fluid was analysed by ICP-AES.

[0155] Statistically significant differences in mass fraction levels of 46 chemical elements (Table 6) were found in seminal fluid samples derived from cancerous, benign hyperplastic and normal subjects. Differences in mass fraction levels of these elements can be used for diagnosis and therapeutic purpose. The data in Table 6 allow evaluating the importance of the individual chemical element content information for the diagnosis of prostate cancer (PCa).

TABLE-US-00006 TABLE 6 Comparison of mean values of chemical element mass fractions (mg .Math. kg.sup.1, dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) seminal fluid. Ratios of means Seminal fluid BPH to PCa to PCa to Element Normal BPH PCa Normal Normal BPH Li 0.04 0.17 0.01 4.6 0.32 0.1 B 0.80 4.19 1.00 5.2 1.25 0.2 Na 21489 42638 35000 2.0 1.63 0.8 Mg 1100 3674 140 8.2 0.13 0.04 Al 3.82 6.06 4.69 1.6 1.23 0.8 Si 6.31 29.35 11.92 4.6 1.89 0.4 P 10352 8453 15000 0.8 1.45 1.8 S 1966 4716 2700 2.4 1.37 0.6 K 5201 21428 3800 4.1 0.73 0.2 Ca 3034 8237 1300 5.0 0.43 0.2 Sc 0.05 0.04 0.01 0.9 0.22 0.2 Cr 0.41 0.55 1.20 1.3 2.91 2.2 Mn 0.23 0.35 0.12 1.6 0.55 0.4 Fe 19.0 17.3 8.5 0.9 0.45 0.5 Co 0.01 0.03 0.01 2.9 1.00 0.3 Ni 0.22 3.27 0.10 14.6 0.45 0.03 Cu 2.16 12.67 0.99 5.9 0.46 0.1 Zn 731 4003 140 5.5 0.19 0.03 Se 0.68 1.41 0.36 2.1 0.54 0.3 Br 30.8 42.5 54.7 1.4 1.78 1.3 Rb 7.0 24.1 5.0 3.4 0.71 0.2 Sr 0.45 2.28 0.50 5.0 1.09 0.2 Y 0.01 0.01 0.01 0.7 0.73 1.0 Zr 0.08 0.08 0.05 1.0 0.59 0.6 Ag 0.01 0.01 0.03 1.0 2.99 3.0 Sb 0.10 0.10 0.01 1.0 0.10 0.1 Cs 0.01 0.06 0.01 4.5 0.99 0.2 Ba 0.13 0.25 0.13 1.9 1.01 0.5 La 0.10 0.01 0.01 0.1 0.10 1.0 Ce 0.01 0.01 0.01 1.0 0.81 0.8 Pr 0.01 0.01 0.01 1.0 1.00 1.0 Nd 0.01 0.01 0.01 1.0 1.00 1.0 Sm 0.01 0.01 0.00 1.0 0.16 0.2 Gd 0.01 0.01 0.01 1.0 1.00 1.0 Tb 0.01 0.01 0.01 1.0 1.00 1.0 Dy 0.01 0.01 0.01 1.0 1.00 1.0 Ho 0.01 0.01 0.01 1.0 1.00 1.0 Er 0.01 0.01 0.01 1.0 1.00 1.0 Au 0.02 0.07 0.01 3.1 0.44 0.1 Cd 0.01 0.03 0.02 2.1 1.58 0.7 Hg 0.04 0.05 0.01 1.4 0.01 0.2 TI 0.01 0.01 0.01 1.0 1.00 1.0 Pb 0.10 0.14 0.08 1.4 0.85 0.6 Bi 0.01 0.01 0.02 0.8 1.27 1.7 Th 0.02 0.01 0.01 0.6 0.56 1.0 U 0.01 0.01 0.01 1.0 1.00 1.0

Example 19

Establishing the Prostate Condition Using Mn Mass Fraction in an EPS Sample

[0156] The tissue content of Mn was found to be significantly different in most cancerous EPS samples as compared to normal and benign hyperplastic EPS samples (Example 17, Table 5). Mass fraction of Mn in EPS of normal prostate was found to be 0.51 mg/kg, in BPH 1.10 mg/kg, and in PCa 3.95 mg/kg on dry mass basis (Table 5). The upper limit for Mn mass fraction in dry EPS from normal or BPH subject was determined to be M+2SD or 2.4 mg/kg on dry mass basis.

[0157] If PCa EPS needs to be discriminated from normal and BPH and if Mn content in the EPS sample prepared and analysed as described in the Example 17 exceeds 2.4 mg/kg dry EPS, prostate carcinoma can be diagnosed with an accuracy of 943%.

Example 20

Establishing the Prostate Condition Using Al Mass Fraction in an EPS Sample

[0158] The tissue content of Al was found to be significantly different in most cancerous EPS samples as compared to normal and benign hyperplastic EPS samples (Example 17, Table 5). Mass fraction of Al in EPS of normal prostate was found to be 4.63 mg/kg, in BPH 12.5 mg/kg, and in PCa 43.85 mg/kg on dry mass basis (Table 5). The upper limit for Al mass fraction in dry EPS from normal or BPH subject was determined to be M+2SD or 25 mg/kg on dry mass basis.

[0159] If EPS PCa needs to be discriminated from normal and BPH and if Al content in a EPS sample prepared and analysed as described in the Example 17 exceeds 25 mg/kg in dry EPS, carcinoma with an accuracy of 964% can be diagnosed.

Example 21

Establishing the Prostate Condition Using Ba Mass Fraction in an EPS Sample

[0160] The tissue content of Ba was found to be significantly different in most cancerous prostatic fluid samples as compared to normal and benign hyperplastic prostatic fluid samples (Example 17, Table 5). Mass fraction of Ba in EPS of normal prostate was found to be 0.35 mg/kg, in BPH 0.35 mg/kg, and in PCa 3.57 mg/kg on dry mass basis (Table 5). The upper limit for Ba mass fraction in dry EPS from normal or BPH subject was determined to be M+2SD or 1.5 mg/kg on dry mass basis.

[0161] If EPS PCa needs to be discriminated from normal and BPH and if Ba content in the EPS sample prepared and analysed as described in the Example 17 exceeds 2.5 mg/kg dry EPS, prostate carcinoma with an accuracy of 955% can be diagnosed.

Example 22

Establishing the Prostate Condition Using Bi Mass Fraction in an EPS Sample

[0162] The tissue content of Bi was found to be significantly different in most cancerous prostatic fluid samples as compared to normal and benign hyperplastic prostatic fluid samples (Example 17, Table 5). Mass fraction of Bi in EPS of normal prostate was found to be 0.02 mg/kg, in BPH 0.01 mg/kg, and in PCa 0.40 mg/kg on dry mass basis (Table 5). The upper limit for Bi mass fraction in dry EPS from normal or BPH subject was determined to be 0.04 mg/kg on dry mass basis.

[0163] If EPS PCa needs to be discriminated from normal and BPH and if Bi content in a seminal fluid sample prepared and analysed as described in the Example 17 exceeds 0.03 mg/kg dry EPS, prostate carcinoma with an accuracy of 963% can be diagnosed.

Example 23

Establishing the Prostate Condition Using Ca Mass Fraction in an EPS Sample

[0164] The tissue content of Ba was found to be significantly different in most cancerous prostatic fluid samples as compared to normal and benign hyperplastic prostatic fluid samples (Example 17, Table 5). Mass fraction of Ca in EPS of normal prostate was found to be 11040 mg/kg, in BPH 10758 mg/kg, and in PCa 3800 mg/kg on dry mass basis (Table 5). The lower limit for Ca mass fraction in dry EPS from normal or BPH subject was determined to be 8000 mg/kg on dry mass basis.

[0165] If EPS PCa needs to be discriminated from normal and BPH and if Ca content in the EPS sample prepared and analyzed as described in the Example 17 does not exceed 2000 mg/kg dry EPS, prostate carcinoma with an accuracy of 955% can be diagnosed.

Example 24

Establishing the Prostate Condition Using Mg Mass Fraction in an EPS Sample

[0166] The tissue content of Mg was found to be significantly different in most cancerous prostatic fluid samples as compared to normal and benign hyperplastic prostatic fluid samples (Example 17, Table 5). Mass fraction of Mg in EPS of normal prostate was found to be 5130 mg/kg, in BPH 4549 mg/kg, and in PCa 1900 mg/kg on dry mass basis (Table 5). The lower limit for Mg mass fraction in dry EPS from normal or BPH subject was determined to be 3500 mg/kg on dry mass basis.

[0167] If EPS PCa needs to be discriminated from normal and BPH and if mg content in the EPS sample prepared and analysed as described in the Example 17 does not exceed 3500 mg/kg dry EPS, prostate carcinoma with an accuracy of 955% can be diagnosed.

Example 25

Establishing the Prostate Condition Using Cr Mass Fraction in a Seminal Fluid Sample

[0168] The tissue content of Cr was found to be significantly different in most cancerous seminal fluid samples as compared to normal and benign hyperplastic seminal fluid samples (Example 18, Table 6). Mass fraction of Cr in seminal fluid of normal prostate was found to be 0.41 mg/kg, in BPH 0.55 mg/kg, and in PCa 1.2 mg/kg on dry mass basis (Table 6). The upper limit for Cr mass fraction in dry seminal fluid from normal or BPH subject was determined to be 0.90 mg/kg on dry mass basis.

[0169] If PCa needs to be discriminated from normal and BPH and if Cr content in a seminal fluid sample prepared and analysed as described in the Example 18 exceeds 0.90 mg/kg dry tissue, prostate carcinoma with an accuracy of 943% can be diagnosed.

Example 26

Establishing the Prostate Condition Using Mg Mass Fraction in a Seminal Fluid Sample

[0170] The tissue content of Mg was found to be significantly different in most cancerous seminal fluid samples as compared to normal and benign hyperplastic seminal fluid samples (Example 18, Table 6). Mass fraction of Mg in seminal fluid of normal prostate was found to be 1100 mg/kg, in BPH 3674 mg/kg, and in PCa 140 mg/kg on dry mass basis (Table 6). The lower limit for Mg mass fraction in seminal fluid from normal or BPH subject was determined to be 700 mg/kg on dry mass basis.

[0171] If PCa needs to be discriminated from normal and BPH and if Mg content in a seminal fluid sample prepared and analysed as described in the Example 18 does not exceed 700 mg/kg dry tissue, prostate carcinoma with an accuracy of 925% can be diagnosed.

Example 26

Establishing the Prostate Condition Using Ca Mass Fraction in a Seminal Fluid Sample

[0172] The tissue content of Ca was found to be significantly different in most cancerous seminal fluid samples as compared to normal and benign hyperplastic seminal fluid samples (Example 18, Table 6). Mass fraction of Ca in seminal fluid of normal prostate was found to be 3030 mg/kg, in BPH 8237 mg/kg, and in PCa 1300 mg/kg on dry mass basis (Table 6). The lower limit for Ca mass fraction in dry seminal fluid from normal or BPH subject was determined to 2200 mg/kg on dry mass basis.

[0173] If PCa needs to be discriminated from normal and BPH and if Ca content in a seminal fluid sample prepared and analysed as described in the Example 18 does not exceed 2200 mg/kg dry tissue, prostate carcinoma with an accuracy of 905% can be diagnosed.

Example 27

Determination of Mass Fraction Levels of 44 Elements Relative to the Mass Fraction of Calcium in Normal, Cancerous and BPH EPS.

[0174] Mass fraction ratios of the elements mentioned in the Example 17 are different in non-cancerous and cancerous EPS and therefore these can be used as prostate tumor biomarkers. In the Table 7 mass fraction ratios of 44 elements relative to mass fraction of calcium are presented. Further, ratios of the mass fraction ratios for EPS from normal, BPH and cancerous subjects are given. The data in the Table 7 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of calcium for the diagnosis of PCa.

TABLE-US-00007 TABLE 7 Means of ratios and their ratios between mean values of mass fraction ratios of Ca to mass fractions of other chemical elements in EPS from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects. Ratios of means Prostatic fluid BPH to PCa to PCa to Element Normal BPH PCa Normal Normal BPH Ca/Li 24530 60481 18046 2.5 0.7 0.3 Ca/B 11328 4233 3800 0.4 0.3 0.9 Ca/Na 0.2 0.2 0.0 0.9 0.1 0.1 Ca/Mg 2.2 2.4 2.0 1.1 0.9 0.8 Ca/Al 2386 861 87 0.4 0.04 0.1 Ca/Si 428 243 35 0.6 0.1 0.1 Ca/P 6.4 2.2 2.1 0.3 0.3 1.0 Ca/S 2.0 2.1 0.6 1.1 0.3 0.3 Ca/K 0.3 0.4 0.2 1.3 0.7 0.5 Ca/Sc 192704 195808 380000 1.0 2.0 1.9 Ca/Cr 18189 11976 160 0.7 0.01 0.01 Ca/Mn 10931 15155 962 1.4 0.09 0.1 Ca/Fe 703 407 10 0.6 0.01 0.03 Ca/Co 327111 236917 44829 0.7 0.1 0.2 Ca/Ni 34350 2350 541 0.1 0.02 0.2 Ca/Cu 1304 788 263 0.6 0.2 0.3 Ca/Zn 1.3 2.1 1.9 1.6 1.5 0.9 Ca/Se 7072 7429 38000 1.1 5.4 5.1 Ca/Br 347 247 7 0.7 0.02 0.03 Ca/Rb 209 333 159 1.6 0.8 0.5 Ca/Sr 4074 4179 1206 1.0 0.3 0.3 Ca/Y 1062560 769711 138346 0.7 0.1 0.2 Ca/Zr 160360 75539 8171 0.5 0.1 0.1 Ca/Ag 425598 28803 4046 0.1 0.01 0.1 Ca/Sb 110400 21303 38000 0.2 0.3 1.8 Ca/Cs 141702 126577 36370 0.9 0.3 0.3 Ca/Ba 31319 30715 1148 1.0 0.04 0.04 Ca/La 199512 549579 84542 2.8 0.4 0.2 Ca/Ce 859311 699707 48008 0.8 0.1 0.1 Ca/Pr 200727 1005734 174020 5.0 0.9 0.2 Ca/Nd 991023 389312 91545 0.4 0.1 0.2 Ca/Sm 1104000 1019233 156230 0.9 0.1 0.2 Ca/Gd 1104000 903780 106736 0.8 0.1 0.1 Ca/Tb 1104000 1075800 263046 1.0 0.2 0.2 Ca/Dy 1104000 1091721 198095 1.0 0.2 0.2 Ca/Ho 1104000 1075800 315152 1.0 0.3 0.3 Ca/Er 1104000 1075800 151557 1.0 0.1 0.1 Ca/Au 154190 57363 380000 0.4 2.5 6.6 Ca/Cd 431048 126876 10313 0.3 0.02 0.1 Ca/Hg 143862 215160 76000000 1.5 528.3 353.2 Ca/Tl 995043 1075800 24762 1.1 0.02 0.02 Ca/Pb 130574 33968 3684 0.3 0.03 0.1 Ca/Bi 671533 1056778 9446 1.6 0.01 0.01 Ca/Th 431082 926283 86428 2.1 0.2 0.1 Ca/U 1104000 1075800 79838 1.0 0.1 0.1

Example 27

Determination of Mass Fraction Levels of 44 Elements Relative to the Mass Fraction of Zinc in Normal, Cancerous and BPH EPS.

[0175] Mass fraction ratios of the elements mentioned in the Example 17 are different in non-cancerous and cancerous EPS and therefore these can be used as prostate tumor biomarkers. In the Table 8 mass fraction ratios of 44 elements relative to mass fraction of zinc are presented. Further, ratios of the mass fraction ratios for EPS from normal, BPH and cancerous subjects are given. The data in the Table 8 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of zinc for the diagnosis of PCa.

TABLE-US-00008 TABLE 8 Means of ratios and their ratios between mean values of mass fraction ratios of Zn to mass fractions of other chemical elements in EPS from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects. Mass Ratios of means fraction Prostatic fluid BPH to PCa to PCa to ratio Normal BPH PCa Normal Normal BPH Zn/Li 19121 28666 9498 1.50 0.50 0.33 Zn/B 8830 2006 2000 0.23 0.23 1.00 Zn/Na 0.2 0.1 0.0 0.56 0.06 0.11 Zn/Mg 1.7 1.1 1.1 0.67 0.63 0.94 Zn/Al 1860.0 407.9 45.6 0.22 0.02 0.11 Zn/Si 333.6 115.0 18.2 0.34 0.05 0.16 Zn/P 5.0 1.1 1.1 0.21 0.22 1.06 Zn/S 1.6 1.0 0.3 0.64 0.20 0.32 Zn/K 0.2 0.2 0.1 0.82 0.46 0.57 Zn/Ca 0.8 0.5 0.5 0.61 0.68 1.11 Zn/Sc 150218 92808 200000 0.62 1.33 2.15 Zn/Cr 14179 5676 84 0.40 0.01 0.01 Zn/Mn 8521 7183 506 0.84 0.06 0.07 Zn/Fe 548.2 193.0 5.4 0.35 0.01 0.03 Zn/Co 254993 112292 23594 0.44 0.09 0.21 Zn/Ni 26777 1114 285 0.04 0.01 0.26 Zn/Cu 1017 374 138 0.37 0.14 0.37 Zn/Se 5513 3521 20000 0.64 3.63 5.68 Zn/Br 270.3 117.2 3.6 0.43 0.01 0.03 Zn/Rb 162.9 157.7 83.8 0.97 0.51 0.53 Zn/Sr 3175 1981 635 0.62 0.20 0.32 Zn/Y 828296 364822 72814 0.44 0.09 0.20 Zn/Zr 125005 35803 4301 0.29 0.03 0.12 Zn/Ag 331766 13652 2129 0.04 0.01 0.16 Zn/Sb 86060 10097 20000 0.12 0.23 1.98 Zn/Cs 110461 59994 19142 0.54 0.17 0.32 Zn/Ba 24414 14558 604 0.60 0.02 0.04 Zn/La 155525 260485 44496 1.67 0.29 0.17 Zn/Ce 669858 331642 25267 0.50 0.04 0.08 Zn/Pr 156473 476691 91590 3.05 0.59 0.19 Zn/Nd 772531 184524 48182 0.24 0.06 0.26 Zn/Sm 860600 483089 82226 0.56 0.10 0.17 Zn/Gd 860600 428367 56177 0.50 0.07 0.13 Zn/Tb 860600 509900 138445 0.59 0.16 0.27 Zn/Dy 860600 517446 104261 0.60 0.12 0.20 Zn/Ho 860600 509900 165869 0.59 0.19 0.33 Zn/Er 860600 509900 79767 0.59 0.09 0.16 Zn/Au 120196 27189 200000 0.23 1.66 7.36 Zn/Cd 336014 60136 5428 0.18 0.02 0.09 Zn/Hg 112145 101980 40000000 0.91 356.68 392.23 Zn/Tl 775665 509900 13033 0.66 0.02 0.03 Zn/Pb 101786 16100 1939 0.16 0.02 0.12 Zn/Bi 523479 500884 4972 0.96 0.01 0.01 Zn/Th 336041 439033 45488 1.31 0.14 0.10 Zn/U 860600 509900 42020 0.59 0.05 0.08

Example 27

Determination of Mass Fraction Levels of 44 Elements Relative to the Mass Fraction of Calcium in Seminal Fluid From Normal, Cancerous and BPH Subjects

[0176] Mass fraction ratios of the elements mentioned in the Example 18 are different in non-cancerous and cancerous seminal fluid and therefore these can be used as prostate tumor biomarkers. In the Table 9 mass fraction ratios of 44 elements relative to mass fraction of calcium are presented. Further, ratios of the mass fraction ratios for seminal fluid from normal, BPH and cancerous subjects are given. The data in the Table 9 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of calcium for the diagnosis of PCa.

TABLE-US-00009 TABLE 9 Means of ratios and their ratios between mean values of mass fraction ratios of Ca to mass fractions of other chemical elements in seminal fluid derived from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects. Mass Ratios of means fraction Seminal fluid BPH to PCa to PCa to ratio Normal BPH PCa Normal Normal BPH Ca/Li 82584 48711 109809 0.6 1.3 2.3 Ca/B 3801 1967 1300 0.5 0.3 0.7 Ca/Na 0.1 0.2 0.04 1.4 0.3 0.2 Ca/Mg 2.8 2.2 9.3 0.8 3.4 4.1 Ca/Al 794 1360 277 1.7 0.3 0.2 Ca/Si 481 281 109 0.6 0.2 0.4 Ca/P 0.3 1.0 0.1 3.3 0.3 0.1 Ca/S 1.5 1.7 0.5 1.1 0.3 0.3 Ca/K 0.6 0.4 0.3 0.7 0.6 0.9 Ca/Sc 65758 196333 130000 3.0 2.0 0.7 Ca/Cr 7331 15114 1079 2.1 0.1 0.1 Ca/Mn 13462 23277 10428 1.7 0.8 0.4 Ca/Fe 160 476 152 3.0 1.0 0.3 Ca/Co 303400 284857 130000 0.9 0.4 0.5 Ca/Ni 13565 2520 13050 0.2 1.0 5.2 Ca/Cu 1403 650 1316 0.5 0.9 2.0 Ca/Zn 4.2 2.1 9.3 0.5 2.2 4.5 Ca/Se 4480 5858 3567 1.3 0.8 0.6 Ca/Br 99 194 24 2.0 0.2 0.1 Ca/Rb 431 342 260 0.8 0.6 0.8 Ca/Sr 6691 3614 2625 0.5 0.4 0.7 Ca/Y 221025 823700 130000 3.7 0.6 0.2 Ca/Zr 39388 109571 28524 2.8 0.7 0.3 Ca/Ag 303400 823700 43458 2.7 0.1 0.1 Ca/Sb 30340 82370 130000 2.7 4.3 1.6 Ca/Cs 219853 131731 95502 0.6 0.4 0.7 Ca/Ba 22721 32499 9663 1.4 0.4 0.3 Ca/La 30340 830622 130000 27.4 4.3 0.2 Ca/Ce 267497 739297 140752 2.8 0.5 0.2 Ca/Pr 303400 823700 130000 2.7 0.4 0.2 Ca/Nd 303400 823700 130000 2.7 0.4 0.2 Ca/Sm 303400 823700 830635 2.7 2.7 1.0 Ca/Gd 303400 823700 130000 2.7 0.4 0.2 Ca/Tb 303400 823700 130000 2.7 0.4 0.2 Ca/Dy 303400 823700 130000 2.7 0.4 0.2 Ca/Ho 303400 823700 130000 2.7 0.4 0.2 Ca/Er 303400 823700 130000 2.7 0.4 0.2 Ca/Au 134556 116232 130000 0.9 1.0 1.1 Ca/Cd 252833 324398 68768 1.3 0.3 0.2 Ca/Hg 84303 164740 130000 2.0 1.5 0.8 Ca/Tl 303400 823700 130000 2.7 0.4 0.2 Ca/Pb 30681 58599 15384 1.9 0.5 0.3 Ca/Bi 232725 823700 78432 3.5 0.3 0.1 Ca/Th 169944 823769 130000 4.8 0.8 0.2 Ca/U 303400 823700 130000 2.7 0.4 0.2

Example 28

Determination of Mass Fraction Levels of 44 Elements Relative to the Mass Fraction of Zinc in Seminal Fluid Derived from Normal, Cancerous and BPH Subjects

[0177] Mass fraction ratios of the elements mentioned in the Example 18 are different in non-cancerous and cancerous seminal fluid and therefore these can be used as prostate tumor biomarkers. In the Table 10 mass fraction ratios of 44 elements relative to mass fraction of zinc are presented. Further, ratios of the mass fraction ratios for normal seminal fluid, BPH and cancerous seminal fluid are given. The data in the Table 10 allow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of calcium for the diagnosis of PCa.

TABLE-US-00010 TABLE 10 Means of ratios and their ratios between mean values of mass fraction ratios of Zn to mass fractions of other chemical elements in seminal fluid derived from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects. Mass Ratios of means fraction Seminal fluid BPH to PCa to PCa to ratio Normal BPH PCa Normal Normal BPH Zn/Li 19898 23655 11826 1.19 0.59 0.50 Zn/B 916 955 140 1.04 0.15 0.15 Zn/Na 0.03 0.1 0.004 2.76 0.12 0.04 Zn/Mg 1.6 1.1 1.0 0.67 0.62 0.92 Zn/Al 191.3 660.2 29.8 3.45 0.16 0.05 Zn/Si 115.8 136.3 11.7 1.18 0.10 0.09 Zn/P 0.1 0.5 0.01 6.70 0.13 0.02 Zn/S 0.4 0.8 0.1 2.28 0.14 0.06 Zn/K 0.1 0.2 0.04 1.33 0.26 0.20 Zn/Ca 0.4 0.5 0.1 1.10 0.24 0.22 Zn/Sc 15844 95342 14000 6.02 0.88 0.15 Zn/Cr 1766 7339 116 4.16 0.07 0.02 Zn/Mn 3243 11303 1123 3.49 0.35 0.10 Zn/Fe 38.5 231.0 16.4 6.00 0.43 0.07 Zn/Co 73100 138331 14000 1.89 0.19 0.10 Zn/Ni 3268 1224 1405 0.37 0.43 1.15 Zn/Cu 338 316 142 0.93 0.42 0.45 Zn/Se 1079.4 2844.5 384.1 2.64 0.36 0.14 Zn/Br 23.8 94.1 2.6 3.96 0.11 0.03 Zn/Rb 103.9 166.2 28.0 1.60 0.27 0.17 Zn/Sr 1612 1755 283 1.09 0.18 0.16 Zn/Y 53253 400000 14000 7.51 0.26 0.04 Zn/Zr 9490 53209 3072 5.61 0.32 0.06 Zn/Ag 73100 400000 4680 5.47 0.06 0.01 Zn/Sb 7310 40000 14000 5.47 1.92 0.35 Zn/Cs 52970 63970 10285 1.21 0.19 0.16 Zn/Ba 5474 15782 1041 2.88 0.19 0.07 Zn/La 7310 403361 14000 55.18 1.92 0.03 Zn/Ce 64450 359013 15158 5.57 0.24 0.04 Zn/Pr 73100 400000 14000 5.47 0.19 0.04 Zn/Nd 73100 400000 14000 5.47 0.19 0.04 Zn/Sm 73100 400000 89453 5.47 1.22 0.22 Zn/Gd 73100 400000 14000 5.47 0.19 0.04 Zn/Tb 73100 400000 14000 5.47 0.19 0.04 Zn/Dy 73100 400000 14000 5.47 0.19 0.04 Zn/Ho 73100 400000 14000 5.47 0.19 0.04 Zn/Er 73100 400000 14000 5.47 0.19 0.04 Zn/Au 32419 56444 14000 1.74 0.43 0.25 Zn/Cd 60917 157532 7406 2.59 0.12 0.05 Zn/Hg 20312 80000 14000 3.94 0.69 0.18 Zn/TI 73100 400000 14000 5.47 0.19 0.04 Zn/Pb 7392 28456 1657 3.85 0.22 0.06 Zn/Bi 56072 400000 8447 7.13 0.15 0.02 Zn/Th 40946 400033 14000 9.77 0.34 0.03 Zn/U 73100 400000 14000 5.47 0.19 0.04

Example 29

Using the Ca/Mn Mass Fraction Ratio in EPS to Establish Prostate Condition

[0178] The Ca/Mn mass fraction ratio in EPS was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS. The upper limit for Ca/Mn mass fraction ratio on dry mass basis in cancerous EPS was determined to be 1900 (Table 7).

[0179] If PCa needs to be discriminated from normal and BPH and if the Ca/Mn ratio in the EPS sample prepared and analysed as described in Example 17 does not exceed 1900, prostate carcinoma with an accuracy exceeding 96% can be diagnosed.

Example 30

Using the Ca/Al Mass Fraction Ratio in Seminal Fluid to Establish Prostate Condition

[0180] The Ca/Al mass fraction ratio in seminal fluid was found to be significantly different in most cancerous seminal fluid as compared to normal and benign hyperplastic seminal fluid. The upper limit for Ca/Al mass fraction ratio on dry mass basis in cancerous seminal fluid was determined to be 290 (Table 9).

[0181] If PCa seminal fluid needs to be discriminated from normal and BPH one and if the Ca/Al ratio in the seminal fluid sample prepared and analysed as described in Example 18 does not exceed 290, prostate carcinoma with an accuracy exceeding 98% can be diagnosed.

Example 31

Using the Zn/Cu Mass Fraction Ratio in EPS to Establish Prostate Condition

[0182] The Zn/Cu mass fraction ratio in EPS was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS. The upper limit for Zn/Cu mass fraction ratio on dry mass basis in cancerous EPS was determined to be 165 (Table 8).

[0183] If PCa EPS needs to be discriminated from normal and BPH EPS and if the Zn/Cu ratio in the EPS sample prepared and analysed as described in Example 17 does not exceed 165, prostate carcinoma with an accuracy of 95% can be diagnosed.

Example 32

Using the Zn/Cu Mass Fraction Ratio in Seminal Fluid to Establish Prostate Condition

[0184] The Zn/Cu mass fraction ratio in seminal fluid was found to be significantly different in most cancerous seminal fluids as compared to normal and benign hyperplastic seminal fluid. The upper limit for Zn/Cu mass fraction ratio on dry mass basis in cancerous seminal fluid was determined to be 155 (Table 10).

[0185] If PCa EPS needs to be discriminated from normal and BPH EPS and if the Zn/Cu ratio in the EPS sample prepared and analysed as described in Example 18 does not exceed 155, prostate carcinoma with an accuracy better than 95% can be diagnosed.

Example 33

Using the Ca*Mg*Zn/Mn*Bi*Se Mass Fraction Ratio Combination in EPS to Establish Prostate Condition

[0186] The Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio in EPS was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS. The lower limit for Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio on dry mass basis in healthy EPS was determined to be 2E8.

[0187] If PCa EPS needs to be discriminated from normal and BPH EPS and if the Ca*Mg*Zn/Mn*Bi*Se ratio in the EPS sample prepared and analysed as described in Example 18 is below 2E8, prostate carcinoma with an accuracy better than 95% can be diagnosed.

Example 34

Using the Ca*Mg*Zn/Mn*Bi*Se Mass Fraction Ratio Combination in Seminal Fluid to Establish Prostate Condition.

[0188] The Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio in seminal fluid was found to be significantly different in most cancerous seminal fluids as compared to normal and benign hyperplastic seminal fluid. The lower limit for Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio on dry mass basis in healthy seminal fluid was determined to be 2E6.

[0189] If PCa seminal fluid needs to be discriminated from normal and BPH seminal fluid and if the Ca*Mg*Zn/Mn*Bi*Se ratio in the seminal fluid sample prepared and analysed as described in Example 19 is below 2E6, prostate carcinoma with an accuracy better than 95% can be diagnosed.

Example 35

Using the Ca/Ba Mass Fraction Ratio to Establish Prostate Condition

[0190] The Ca/Ba mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Ba mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 400 (FIG. 13).

[0191] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/Ba ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 400, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the Ca/Ba ratio based test is 100-9% and 100-2%, respectively.

Example 36

Using the Ca/P Mass Fraction Ratio to Establish Prostate Condition

[0192] The Ca/P mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/P mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 0.15 (FIG. 14).

[0193] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/P ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 0.15, prostate carcinoma with an accuracy of 982% can be diagnosed. The sensitivity and specificity of the Ca/P ratio based test is 919% and 100-3%, respectively.

Example 37

Using the Ca/Si Mass Fraction Ratio to Establish Prostate Condition

[0194] The Ca/Si mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Si mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 5 (FIG. 15).

[0195] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/Si ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 5, prostate carcinoma with an accuracy of 982% can be diagnosed. The sensitivity and specificity of the Ca/Si ratio based test is 919% and 100-3%, respectively.

Example 38

Using the Ca/Sr Mass Fraction Ratio to Establish Prostate Condition

[0196] The Ca/Sr mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Sr mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 250 (FIG. 16).

[0197] If PCa needs to be discriminated from normal and BPH tissue and if the Ca/Sr ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 250, prostate carcinoma with an accuracy of 982% can be diagnosed. The sensitivity and specificity of the Ca/Sr ratio based test is 919% and 100-3%, respectively.

Example 39

Using the Zn/Mn Mass Fraction Ratio to Establish Prostate Condition

[0198] The Zn/Mn mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 8, Table 2). The upper limit for Zn/Mn mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (Marithmetic mean, SDstandard deviation) or 170 (FIG. 17).

[0199] If PCa needs to be discriminated from normal and BPH tissue and if the Zn/Mn ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 170, prostate carcinoma with an accuracy of 982% can be diagnosed. The sensitivity and specificity of the Zn/Mn ratio based test is 919% and 100-3%, respectively.

Example 40

Using the [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 Mass Fraction Ratio Combination to Establish Prostate Condition

[0200] The [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination on dry mass basis in cancerous prostate tissue was determined to be M+60SD (Marithmetic mean, SDstandard deviation) or 100000 (FIG. 18).

[0201] If PCa needs to be discriminated from normal and BPH tissue and if the [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 100000, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination based test is 100-10% and 100-3%, respectively.

Example 41

Using the Normalized Mass Fraction Ratio Combinations of Ag, Al, Ba,Bi, Br,Ca, Cd, Ce, Co, Cr, Cs, Cu, Hg, K, Li, Mg, Mn, Na, Ni, P, Pb, Rb, S, Sb, Se, Si, Sr and Zn to Establish Prostate Condition from the Prostate Tissue Samples

[0202] Mass fraction levels of the elements can be normalized to the reference levels of same elements. Further, combination of normalized mass fraction ratios can be used to diagnose prostate condition. To illustrate this the normalized mass fraction levels for 27 elements were calculated as mass fraction of the element divided by the median value of the mass fraction of the same element in the tissue samples taken from normal individuals.

[0203] The following combination of normalised mass fraction ratios [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*L i.sub.n*Mn.sub.n*Ni.sub.n*Pb.sub.n*Sb.sub.n*Si.sub.n*Sr.sub.n)]*10.sup.18 was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*L i.sub.n*Mn.sub.n*Ni.sub.n*Pb.sub.n*Sb.sub.n*Si.sub.n*Sr.sub.n)]*10.sup.18 combination of normalised mass fraction ratios on dry mass basis in cancerous prostate tissue was determined to be 100000000000 (FIG. 19).

[0204] If PCa needs to be discriminated from normal and BPH tissue and if the [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*L i.sub.n*Mn.sub.n*Ni.sub.n*Pb.sub.n*Sb.sub.n*Si.sub.n*Sr.sub.n)]*10.sup.18 ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 100000000000, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*L i.sub.n*Mn.sub.n*Ni.sub.n*Pb.sub.n*Sb.sub.n*Si.sub.n*Sr.sub.n)]*10.sup.18 ratio based test is 100-10% and 100-3%, respectively.

Example 42

Using the Normalized Mass Fraction Ratio Combinations of Ag, Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Th, Tl, U, Y, Zn and Zr to Establish Prostate Condition from the Prostate Tissue Samples

[0205] Mass fraction levels of the elements can be normalized to the reference levels of same elements. Further, combination of normalized mass fraction ratios can be used to diagnose prostate condition. To improve the diagnostic value of the normalized mass fraction ratio prostate cancer test the number of elements in the combination can be increased. To illustrate this the normalized mass fraction levels for 45 elements were calculated as mass fraction of the element taken from the list Ag, Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Th, Tl, U, Y, Zn and Zr and divided by the median value of the mass fraction of the same element in the tissue samples taken from normal individuals.

[0206] The [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Sc.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Au.sub.n*B.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*Dy.sub.n*Er.sub.n*Fe.sub.n*Gd.sub.n*Ho.sub.n*La.sub.n*Li.sub.n*Mn.sub.n*Nd.sub.n*Ni.sub.n*Pb.sub.n*Pr.sub.n*Sb.sub.n*Si.sub.n*Sm.sub.n*Sr.sub.n*Th.sub.n*Ti.sub.n*U.sub.n*Y.sub.n *Zr.sub.n)]*10.sup.34 mass fraction ratio combination was found to be significantly different in most cancerous prostate tissue samples as compared to normal and benign hyperplastic tissue samples. The diagnostic window, i.e. the gap between the lowest normalized mass fraction ratio combination from BPH group and the highest normalized mass fraction ratio combination from the prostate cancer group, has increased to five orders of magnitude (FIG. 20). The upper limit for [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Sc.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Au.sub.n*B.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*Dy.sub.n*Er.sub.n*Fe.sub.n*Gd.sub.n*Ho.sub.n*La.sub.n*Li.sub.n*Mn.sub.n*Nd.sub.n*Ni.sub.n*Pb.sub.n*Pr.sub.n*Sb.sub.n*Si.sub.n*Sm.sub.n*Sr.sub.n*Th.sub.n*Ti.sub.n*U.sub.n*Y.sub.n *Zr.sub.n)]*10.sup.34 normalized mass fraction ratio combination on dry mass basis in cancerous prostate tissue was determined to be 10.sup.24 (FIG. 20).

[0207] If PCa needs to be discriminated from normal and BPH tissue and if the [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Sc.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Au.sub.n*B.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*Dy.sub.n*Er.sub.n*Fe.sub.n*Gd.sub.n*Ho.sub.n*La.sub.n*Li.sub.n*Mn.sub.n*Nd.sub.n*Ni.sub.n*Pb.sub.n*Pr.sub.n*Sb.sub.n*Si.sub.n*Sm.sub.n*Sr.sub.n*Th.sub.n*Ti.sub.n*U.sub.n*Y.sub.n *Zr.sub.n)]*10.sup.34 normalised mass fraction ratio combination in a prostate biopsy sample prepared and analyzed as described in Example 1 does not exceed 10.sup.24, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the [(Ca.sub.n*Cd.sub.n*Co.sub.n*Hg.sub.n*K.sub.n*Mg.sub.n*Na.sub.n*P.sub.n*Rb.sub.n*S.sub.n*Sc.sub.n*Se.sub.n*Zn.sub.n)/(Ag.sub.n*Al.sub.n*Au.sub.n*B.sub.n*Ba.sub.n*Bi.sub.n*Br.sub.n*Ce.sub.n*Cr.sub.n*Cs.sub.n*Cu.sub.n*Dy.sub.n*Er.sub.n*Fe.sub.n*Gd.sub.n*Ho.sub.n*La.sub.n*Li.sub.n*Mn.sub.n*Nd.sub.n*Ni.sub.n*Pb.sub.n*Pr.sub.n*Sb.sub.n*Si.sub.n*Sm.sub.n*Sr.sub.n*Th.sub.n*Ti.sub.n*U.sub.n*Y.sub.n *Zr.sub.n)]*10.sup.34 normalized mass fraction ratio combination based test is 100-10% and 100-3%, respectively.

Example 43

Identification of Cancer Biomarkes in Expressed Prostatic Secretion Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Atomic Emission Spectrometry (ICP-AES).

[0208] Equipment:

[0209] Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

[0210] Specimen:

[0211] Expressed Prostatic Secretion samples (EPS) from patients with Benign Prostate Hyperplasia (BPH) and low-grade prostate adenocarcinoma (PCa) and EPS samples from healthy volunteers were obtained by transrectal prostate massage. The presence or absence of cancer was confirmed by Digital Rectal Examination (DRE), TransRectal Ultrasound Imaging (TRUSI) and microscopic analysis of tissue morphology in biopsies obtained from the same patients, where prescribed by the referring physician.

[0212] Reagents:

[0213] HNO3 (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H.sub.2O.sub.2 (hydrogen peroxide pure for analysis, Merck), ICP-MS standards NCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureau of Reference of the European Comission) and IRMBD151 (LGC Standards, Weisel, Germany).

[0214] Protocol:

[0215] 0.5 mL of HNO.sub.3 was added to freeze-dried EPS samples and the samples were left over night at room temperature. After that 0.25 mL of HNO.sub.3 and 0.15 mL of H.sub.2O.sub.2 were added to the samples and placed in water bath at 95 C. for 30 min. The heat-treated samples were cooled down to the room temperature; the soluble fraction was diluted with deionized water to 15 mL and transferred to a plastic measuring bottle. Simultaneously, the same procedure was performed on a sample containing no EPS fluid, and the resultant solution was used as a blank sample. All samples were analyzed by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

[0216] The spectrometer parameters and the main parameters of ICP-MS measurements: auxiliary air flow rate0.9 L/min, nebulizer flow rate0.9 L/min, sample update0.8 mL/min. The spectrometer parameters for ICP-AES measurements: generator output power 1,500 W.

[0217] Results:

[0218] The content of Al, Cd, Cs, Mn, Ni, Rb, S, Se and Si in EPS was analyzed by ICP-MS. The content of Na, Mg, P, S, K, Ca, Fe, Cu, Zn and Ba in EPS was analyzed by ICP-AES.

[0219] Statistically significant differences in mass fraction levels of 18 chemical elements (Table 11) were found in samples derived from low grade cancerous, benign hyperplastic and normal EPS.

[0220] Differences in mass fraction levels of these elements can be used for diagnosis and therapeutic purpose. The data in Table 5 allow evaluating the importance of the individual chemical element content information for the diagnosis of clinical prostate cancer (PCa).

TABLE-US-00011 TABLE 11 Comparison of median values of chemical element mass fractions (mg .Math. kg.sup.1, dry mass basis) in normal, benign hyperplastic (BPH) and low grade cancerous (PCa) EPS. Normal BPH PCa BPH/Normal PCa/Normal PCa/BPH Al 29.20 13.06 91.21 0.4 3.1 7.0 Ba 1.12 0.42 3.23 0.4 2.9 7.8 Ca 9989 9729 14000 1.0 1.4 1.4 Cd 0.04 0.04 0.03 1.0 0.9 0.9 Cs 0.10 0.08 0.09 0.9 0.9 1.1 Cu 6.24 5.27 5.83 0.8 0.9 1.1 Fe 25.68 27.54 24.38 1.1 0.9 0.9 K 33500 29367 48000 0.9 1.4 1.6 Mg 4644 4549 6500 1.0 1.4 1.4 Mn 0.50 0.93 1.58 1.8 3.1 1.7 Na 41286 51010 47000 1.2 1.1 0.9 Ni 0.43 0.83 0.73 1.9 1.7 0.9 P 3350 3800 4400 1.1 1.3 1.2 Rb 40.75 30.93 47.66 0.8 1.2 1.5 S 5809 6400 10521 1.1 1.8 1.6 Se 1.24 1.26 1.97 1.0 1.6 1.6 Si 84.32 105.4 110.0 1.3 1.3 1.0 Zn 5135 4100 8000 0.8 1.6 2.0

Example 44

Determination of Normalized Mass Fraction Levels of Elements in Normal, BPH and Low Grade Adenocarcinoma EPS

[0221] Mass fraction levels of the elements can be normalized to the reference levels of same elements. In the Table 12 mass fraction ratios of 18 elements relative to reference levels of the same elements are presented. Reference levels in this example represent mean level values derived from the group of 10 EPS samples from verified healthy volunteers. Anybody skilled in the field can appreciate that corresponding reference levels must be determined for different patient populations.

TABLE-US-00012 TABLE 12 Mean mass fraction levels of elements normalized to the reference levels of the same elements in normal, BPH and low grade adenocarcinomatous EPS. Example Reference levels, mg/kg dry mass BPH/ PCa/ PCa/ basis Normal BPH Pca Normal Normal BPH Al 33.89 0.9 1.1 2.6 1.1 2.6 2.4 Ba 4.27 1.0 1.1 3.3 1.1 3.3 3.1 Ca 9528 1.1 1.0 1.5 1.0 1.5 1.5 Cd 0.13 0.9 0.7 0.5 0.7 0.5 0.8 Cs 0.12 0.9 0.7 1.1 0.7 1.1 1.6 Cu 7.31 1.0 1.2 1.3 1.2 1.3 1.1 Fe 44.66 0.9 1.4 1.0 1.4 1.0 0.8 K 32402 1.1 1.0 1.6 1.0 1.6 1.7 Mg 4225 1.1 1.0 1.6 1.0 1.6 1.5 Mn 0.94 1.0 1.8 1.5 1.8 1.5 0.8 Na 39362 1.0 1.5 1.5 1.5 1.5 1.0 Ni 0.74 0.9 2.6 1.5 2.6 1.5 0.6 P 3901 0.9 1.1 1.0 1.1 1.0 0.9 Rb 40.15 1.1 0.8 1.4 0.8 1.4 1.8 S 6471 1.0 1.1 1.6 1.1 1.6 1.5 Se 1.52 1.0 0.9 1.4 0.9 1.4 1.6 Si 75.41 0.9 1.6 2.6 1.6 2.6 1.6 Zn 4870 1.0 0.8 1.7 0.8 1.7 2.1

[0222] The data in the Table 12 allow evaluating the importance of normalized mass fraction levels for the diagnosis of PCa. To illustrate this further examples are given.

Example 45

Establishing the Prostate Condition Using the Additive Index Based on the Normalized Mass Fractions of Ca, K, Mg and Zn in EPS

[0223] Further, based on the normalized mass fractions of the elements determined as described in Example 44 the Additive Index (AI) of prostate condition can be calculated, as exemplified here:

AI=(Ca.sub.n+K.sub.n+Mg.sub.n+Zn.sub.n)4

where Ca.sub.n, K.sub.n, Mg.sub.n, Zn.sub.n represent normalized values, i.e. mass fractions of Ca, K, Mg and Zn in EPS samples of the subject, divided by the reference levels of the same elements. Additive Index was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS (Table 13).

[0224] If PCa needs to be discriminated from normal and BPH and if the Additive Index in the EPS sample prepared and analyzed as described in Example 43 exceeds the value of 1.0, prostate carcinoma with an accuracy exceeding 95% can be diagnosed (FIG. 21).

TABLE-US-00013 TABLE 13 Comparison of the Additive Indices between the diagnostic groups. Normal BPH PCa Mean -3e-007 -0.21 2.0 Std. Deviation 1.2 1.0 1.1 Std. Error of Mean 0.39 0.28 0.49 Lower 95% Cl of mean -0.9 -0.8 0.6 Upper 95% Cl of mean 0.9 0.4 3.3

Example 46

Establishing the Prostate Condition Using the Multiplicative Index Based on the Normalized Mass Fractions of Ca, K, Mg and Zn in EPS

[0225] Further, based on the normalized mass fractions of the elements determined as described in Example 44 the Multiplicative Index (MI) of prostate condition can be calculated, as exemplified here:

MI=(Ca.sub.n*K.sub.n*Mg.sub.n*Zn.sub.n)/4

where Ca.sub.n ,K.sub.n,Mg.sub.n,Zn.sub.n represent mass fractions of Ca, K, Mg and Zn in EPS of the subject normalised to the reference levels. Multiplicative Index was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS (Table 14).

[0226] If PCa needs to be discriminated from normal and BPH and if the Multiplicative Index in the EPS sample prepared and analysed as described in Example 43 exceeds the value of 0.7, prostate carcinoma with an accuracy exceeding 99% can be diagnosed (FIG. 22).

TABLE-US-00014 TABLE 14 Comparison of the Multiplicative Indices between the diagnostic groups. Normal BPH PCa Mean 0.4 0.2 1.8 Std. Deviation 0.27 0.16 1.3 Std. Error of Mean 0.08 0.04 0.6 Lower 95% Cl of mean 0.2 0.1 0.1 Upper 95% Cl of mean 0.5 0.3 3.5

Example 47

Establishing the Prostate Condition Using the Multiplicative Index Based on the Normalized Mass Fractions of Ca, K, Mg, Rb, S and Zn in EPS

[0227] Further, based on the normalized mass fractions of the elements determined as described in Example 44 the 6-element Multiplicative Index (MI/6) of prostate condition can be calculated, as exemplified here:

MI/6=(Ca.sub.n*K.sub.n*Mg.sub.n*Rb.sub.n*S.sub.n*Zn.sub.n)/6

where Ca.sub.n,K.sub.n,Mg.sub.n, Rb.sub.n,S.sub.n and Zn.sub.n represent mass fractions of Ca, K, Mg, Rb, S and Zn in EPS of the subject normalised to the reference levels. Multiplicative Index was found to be significantly different in most cancerous EPS as compared to normal and benign hyperplastic EPS (Table 15).

[0228] If PCa needs to be discriminated from normal and BPH and if the Multiplicative Index in the EPS sample prepared and analysed as described in Example 43 exceeds the value of 0.9, prostate carcinoma with an accuracy exceeding 95% can be diagnosed (FIG. 23).

TABLE-US-00015 TABLE 15 Comparison of the Multiplicative Indices between the diagnostic groups. Normal BPH PCa Mean 0.3 0.2 4.1 Std. Deviation 0.3 0.13 6.4 Std. Error of Mean 0.1 0.04 2.9 Lower 95% Cl of mean 0.13 0.07 -3.9 Upper 95% Cl of mean 0.6 0.2 12

Example 48

Identification of Cancer Biomarkes in Expressed Prostatic Secretion Using Energy Dispersive X-Ray Fluorescence (EDXRF)

[0229] Equipment and Method:

[0230] EDXRF spectrometer consisted of an annular .sup.109Cd source with an activity of 2.56 GBq, a 25 mm.sup.2 Si(Li) detector and portable multichannel analyzer combined with a PC. Its resolution was 270 eV at the 5.9 keV line of 55Fe-source. The duration of the Zn measurements together with Br, Fe, Rb, and Sr was 60 min. The intensity of K-line of Br, Fe, Rb, Sr, and Zn for samples and standards was estimated on the basis of calculating the total area of the corresponding photopeak in the spectra. The element content was calculated by comparing intensities of K-lines for samples and standards.

[0231] Specimen:

[0232] Expressed Prostatic Secretion samples (EPS) from patients with Benign Prostate Hyperplasia (BPH) and adenocarcinoma (PCa) and EPS samples from healthy volunteers were obtained by transrectal prostate massage. The presence or absence of cancer was confirmed by Digital Rectal Examination (DRE), Ultrasound Imaging (TRUSI) and microscopic analysis of tissue morphology in biopsies obtained from the same patients, where prescribed by the referring physician.

[0233] Sample Preparation:

[0234] 20 l of the EPS sample were placed on a backing comprised of a thin film of transparent polymeric material (Dacron, Mylar, polyethylene or similar, thickness<10 m). The drop of a sample was freeze-dried on a backing until the constant mass.

[0235] Results:

[0236] The content of Zn, Br, Fe, Rb, and Sr in EPS obtained from 32 healthy volunteers, 23 BPH patients and 10 prostate adenocarcinoma patients was analyzed by EDXRF.

[0237] Differences in mass fraction levels of Zn and Rb were found to be statistically significant in samples derived from cancerous, benign hyperplastic and normal EPS samples.

[0238] Combination of these elements can be used for diagnosis and therapeutic purpose. The product of mass fraction levels of Rb and Zn divided by ten, as expressed by the following formula: (Rb*Zn)/10 was found to be the most informative marker of prostate cancer. The data in Table 16 allow evaluating the importance of the combination of mass fraction levels of Rb and Zn for the diagnosis of clinical prostate cancer (PCa).

[0239] If PCa needs to be discriminated from normal and BPH and if the Product index (Rb*Zn)/10 in the EPS sample prepared and analysed as described in Example 48 exceeds the value of 350, prostate carcinoma with an accuracy exceeding 98% can be diagnosed (FIG. 24).

TABLE-US-00016 TABLE 16 Parameters of the importance (sensitivity, specificity and accuracy) of the Product index (Rb Zn)/10 in the samples of expressed prostatic secretion for the diagnosis of PCa (an estimation is made for PCa or Intact and BPH). Upper limit for PCa Sensitivity, % Specificity, % Accuracy, % <350 100-10 100-2 100-2