METHOD OF DIAGNOSING PROSTATE CANCER BY DETECTING CHEMICAL

ELEMENTS

FIELD OF THE INVENTION

The present invention relates to a sensitive diagnostic method for establishing a prostate condition.

BACKGROUND OF THE INVENTION Description of the related art

Prostate cancer (PCa) remains the second most common cancer worldwide for maies with an estimated 900,000 new cases diagnosed in 2008 (Ferlay J, et aL Estimates of cancer incidence and mortality in Europe in 2008, European Journal of Cancer, 2010 46:765 -781 ). According to the Cancer Research UK, PCa is the most common cancer in males in the UK, accounting for 41 ,000 of new cases of cancer in maies every year. In 2008-2010 26% of PCa cases in the UK are diagnosed in men under the age of 65 (CancerStafs, incidence 2009 - UK, CRUK May 2012).

Prostate cancer normally causes no symptoms until the cancer has grown large enough to put pressure on the urethra. Symptoms can inciude weak urinal fiow, frequent urination, pain when passing urine etc. Due to the fact that benign prostate conditions such as inflammation, infection and benign prostatic hyperplasia are common in men over the age of 50 and produce similar symptoms, discrimination between prostate cancer and benign prostatic conditions presents a challenge to current diagnostic methods. Currently, there is no single, effective screening test to accurately diagnose prostate cancer in men. The most commonly used PCa diagnostic methods today include the serum prostate-specific antigen analysis {PSA), the digital rectal examination (DRA), and the ultrasound-guided prostate biopsy sampling {Harwich A, et al. Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 21 (Supplement 5): v129~y133, 2010). Despite the years of research the specificity and sensitivity of the PSA based multi-step diagnostic approach is still highly inaccurate. For example, in the European Randomized Study 75.9% of men who underwent a biops because of an elevated PSA value had no cancer {Schroeder F, et ai. Screening and Prostate-Cancer Mortality in a Randomized European Study, N Engl J Med 2009; 360:1320), In addition, a needle biopsy is an invasive and painful procedure with side effects such as prostatitis and blood In urine or semen. Also, many men find the DRA and the needle biopsy embarrassing, in addition to high level of false-positive results, smaller tumors can be missed by current methods with fata! consequences because prostate tumors have the potential to suddenly grow and metastasize.

There is constant search for novel biomarkers to improve specificity of PCa detection. For example, one of such biomarkers currently under clinical investigations is the prostate specific non-coding mRNA marker. PCA3, measured in urine sediment obtained after prostatic massage {Heidenreich A, et ai. Guideiines on Prostate Cancer, European Association of Uroiogy 2010). So far, however, none of the investigational biomarkers are being used routinely.

Prostate tissue (Zaichick S and Zaichick V, IIMAA application in the age dynamics assessment of Br, Ca, CI, K, Mg, Mn, and Na content in the normal human prostate. J Radioanai Nucl Chem 2011; .288:197-202), expressed prostatic secretions (EPS) (Costelio L and Frankiin R. Prostatic fluid ^■ electrolyte composition for the screening of prostate cancer: a potential solution to a majo problem. Prostate Cancer Prostatic Dis 2008;12(1 ): 17-24) and seminal fluid (Owen D. and Katz D, A Review of the Physical and Chemical Properties of Human Semen and th Formulation of a Semen Simulant. J Androl 2005;26: 459-469) contain unusually high amounts of electrolytes such as K, Na, Zn, Ca, Mg, CI, Br and others. The reason for the unusually high metal ion content in normal prostate gland and its excretions is not completely understood, but it was shown that decrease in zinc levels in prostate tissue (Zaichick V, et ai. SU997281 ), prostatic fluid (Zaichick V, et !. Zinc concentration in human prostatic fluid: normal, chronic prostatitis, adenoma and cancer. int Urol Nephroi 1996;28(5): 687-694) and seminal fluid (Frederiekson C. US 2004/229300 AI and US 2010/0099 95 A!; and Leslie C. Costelio and Renty B. Franklin, US 20 1/0046204 AI ) can be used to indicate the risk of prostate cancer. Until now this method has not found practical application.

Thus, so far no reliable method has been developed for prostate cancer detection. Therefore there is a need for a rapid and non-invasive routine prostate cancer test, which can detect PCa in asymptomatic men or discriminate between benig and malignant prostatic conditions in patients with prostatic symptoms.

SUMMARY OF THE INVENTION

The invention provides a method of diagnosing a prostate condition, as defined in the independent claims, to which reference should now be made. Advantageous or preferred features are set forth in dependent claims. According to an aspect of the invention, there may be provided a method of diagnosing a prostate condition in a subject, comprising:

determining, in a sample obtained from a subject, a Ievei of at least one constituent selected from the group consisting of Ag, Ai, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, g, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, T!, U, Y, Zn and Zr; and

comparing the ievei 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 ievei of the same at ieast one constituent, is indicative of the prostate condition.

In another aspect of the invention, there may be provided a method of diagnosing a prostate condition in a subject, comprising:

determining, in a sample obtained from a subject, a level of at least one constituent (or first constituent) selected from the group consisting 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, Se, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, Zn and Zr; and either

(a) comparing a combination of levels of a plurality of constituents from the at Ieast one constituent in the sample (i.e. a sample combination) with a combination of control levels of the same plurality of constituents (i.e. a control combination), or

(b) determining a level of at Ieast one further constituent (or second constituent) not selected from the group consisting 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, Se, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, Zn and Zr, and

comparing a combination of the level of the at Ieast one constituent and the level of the at least one further constituent in the sample (i.e. a sample combination) with a combination of control levels of the same at least one constituent and the same at ieast one further constituent (i.e. a control combination),

In which a difference between the combinations is indicative of the prostate condition. ln a preferred embodiment, the sampie is, or comprises, a bodily fiuid. The bodiiy fluid may be blood, blood plasma, urine, prostatic fluid, expressed prostatic secretion or seminal fluid. Preferabiy, the bodiiy fiuid comprises expressed prostatic secretion or seminal fluid. Aiternativeiy, the sampie may be, or may comprise, a bodiiy tissue such as prostate tissue. The prostate tissue may be obtained by biopsy.

The at Ieast one constituent may be selected from the group consisting of Ca, K, Mg, Ag, Ai, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, n, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y and Zr. This group may be particularly preferabie, should the sampie be a bodiiy fluid.

In anothe preferred embodiment, the at ieast one constituent is selected from the group consisting of Ai, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn, Se, Tb, Th, U,Y and Zn, or is seiected from the group consisting of AI, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn, Se, Tb, Th, U and Y. Either of these groups may be particuiar!y preferable, should the sampie be a bodiiy fluid.

In an aifernative embodiment the at ieast one constituent is selected from the group consisting of Mn, AI, Ba, 8i, Ca, Mg, K, Se and Cr. This group may be particularly preferabie, should the sample be a bodily fluid.

The at Ieast one constituent may be selected from the group consisting of Ca, K, Mg, Ai. Au, B, Ba, Bi, Br, Cd, Ce, Cs, Dy, Er, Gd, Ho, La, LI, Na, Nd, Ni, P, Pb, Pr, S, Si, Sm, Sr, Tb, Th, Tl, U, Y and Zr. This group may be particularly preferabie, should the sampie be a tissue sampie. in another preferred embodiment, the at ieast one constituent, is selected from the group consisting ^■ of AI, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn, Se and Zn, or is seiected from the group consisting of Ai, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn and Se. Either of these groups may be particuiariy preferabie, should the sample be a tissue sample. in an aiiernaiive embodiment, the at ieast one constituent is seiected from the grou consisting of AI, Ba, Bi, Ca, Mg and Mn. This group may be particuiariy preferable, should the sample be a tissue sampie. In an alternative embodiment the at least one constituent is selected from the group consisting of Ai, Ba, Bi, Ca, Cd, Cu, Fe, Mg, Mn and Ni. The combination of constituents may comprise determining one or more ratios. For example, the method may comprise determining a ratio between a first constituent and a second constituent, or between two or more constituents, selected from the group consisting of Ag, A!, Au, B, Ba, Bi, Br. Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, n, Na, Nd, Ni, P, Pb, Pr, Rh, S, Sfa, Sc, Se, Si, Sm, Sr, Tb, Th, Ti, U, Y, Zn and Zr.

In preferred embodiments, the method may comprise determining a ratio of a constituent in relation to Ca, or in relation to Zn. Preferred ratios may include Ca/Ba, Ca/Fe, iVig/AI, Ca/Cu, Mg/Cu, Zn Cu, Zn/Mn, Ca/ n, Ca/P, Ca Si, Ca Sr, or Ca/AS.

Assessing combinations of constituents may comprise comparing relationships betwee ratios of constituents. For example, a first sample ratio may be calculated between a first constituent and a second constituent. A second sample ratio may be calculated between a first constituent (which may be the same or different from the first constituent of the first ratio) and a second constituent (which may be the same or different from the second constituent of the first ratio). Either the first or second constituent of the second ratio will thus be different from the first ratio. For example, relationships between ratios may include multiples of two or more ratios such as (Ca/Cu) ^* (Mg/Cu) (Ca/Cu)*(Zn Cu); or(Mg/Cu)*(Zn/Cu). Such relationships may then be compared with relationships between control ratios of the same constituents.

Combinations of constituents may include ratios between multiples of two or more constituents. As an illustration, this ma include (Ca ^* Mg*Zn) (A[*Bi*Cu). (Ca ^' * g*Zn)/(Mn*8i ^* Se) or (Zn*Ca*Mg*Cd)/(Si ^* Br*Ai*Ba) .

Combinations of constituents may comprise multiplication of levels of two or more constituents. As an illustration, this may include (Zn*Rb)/10.

In another preferred embodiment, comparing a combination of levels of constituents with a combination of control levels of the same constituents may involve normalized levels of constituents. For example, constituents may be normalized to control or reference levels of the same constituents. For instance, normalization may include dividing a level of constituent with a average {such as a median or a mean) level of the same constituent taken from norma! individuals. A normalized amount of a constituent, such as a normalized mass fraction of an element, may be represented by _n - Combinations of normalized ieve!s may be used. As an illustration, ibis may include

!n yet another preferred embodiment, combinations of normalized ieveis of constituents may be used. As an iiiusiration this may include the use of multiplicative indices, such as {Caa*Kn ^* Mgn.*Rbn*Sn:*Zn _n )/6- or (Ca„*Kn*Mg„*Zn _n } 4. This combination may be particularly preferable, should the sample be a bodily fluid.

In another aspect of the invention, combinations of normalized levels of constituents may represent the sum of normalized ieveis, such as no nalized mass fractions. As an Iiiusiration an additive index, such as (Can+ _n ⁺ Mgn ⁺ Znn)-4, may be used. This additive combination may be particu!ariy preferable, shou!d the sample be an expressed prostatic secretion. in an embodiment of the invention, the method may inciude the step of obtaining sample from a subject. Determination of levels of constituents in samples from a subject may occur ex vivo or in vitro, The at least one further (or second) constituent which is not selected from the group consisting of Ag, Ai, AM, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Out, Dy, Er, Fe, Gc!, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Ti, U, Y, Zn and Zr, may be any chemical eiement or any chemical substance such as a protein, DNA or RNA, or any other gene derived product.

The prostate conditio may be benign prostatic hyperplasia. Preferably, the prostate condition is prostate cancer.

In another aspect of the Invention, there may be provided a device for carrying out a method; the method as described in any form above.

One aim of the present invention may be to provide a rapid, specific, non-invasive and sensitive diagnostic method of establishing the condition of prostatic gland, in particular early non -invasive detection of prostate cancer. in a broad sense, the method is based on determination of the ieveis of certain chemical elements present in a biologicai sample from a subject to establish the prostate condition. The obtained levels and/or any ratio between at least one of the obtained levels and the level of any chemical element or any chemical substance such as a protein, DNA or RNA, or any other gene derived product present in the biological sample from the same subject, and/or any combination of said ratios or said levels may be compared to control levels, ratio of the control levels or their combination. Differentia! presence of the said biomarkers as compared to the control ma be indicative of the prostate condition.

In one aspect, there ma be provided a device for defection of the levels of certain chemical elements as biomarkers in a ioSogtcai sample to establish the prostate condition. The obtained levels and/or any ratio between at least one of the obtained levels and the level of any chemical element, chemical substance or protein in the biological sample from the same subject, and/or any combination of said ratios or said levels may be compared to control levels, ratio of the control levels or their combination. Differential presence of the said biomarkers as compared to the control may be indicative of the prostate condition. In another aspect, there may be provided the use of determination of the levels of certain chemical elements as biomarkers in a biologicai sample for establishing the prostate condition. The obtained levels and/or any ratio between at least one of the obtained levels and the ievei of any chemicai element, chemical substance or protein in the biologicai sample from the same subject, and/or any combination of said ratios or said levels may be compared to control levels, ratio of the control !eve!s or their combination. Differential presence of the said biomarkers as compared to the control may be indicative of the prostate condition.

Comparing a level of a constituent with a control level of the constituent, or a combination of levels of constituents with a combination of control levels of the constituents may provide an indication of the presence or absence of a prostate condition. The method may also relate to a devic or tool to establish the prostate condition.

Definitions

As used herein, the term, "a" or "an" may mean one or more. As used herein in the elaim(s), whe used in conjunction with the word "comprising", the word "a" or "an" may mean one but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein. As used herein "another" or "other" may mean at least a second or more of the same or different claim element or components thereof.

"Biornarker" means a chemical element that, is differentially present (i.e., increased or decreased) in a biological sampie from a subject or a group of subjects having a first phenotype {e.g., having a disease) as compared to a biological sampie from a subject or group of subjects having a second phenotype (e.g., not having the disease). A biornarker is preferably differentially present at a level that is statistically significant (i.e., a p-value iess tha 0.05 and/or a q-value of iess than 0,10 as determined using either Welch's T~test or Wilcoxon's rank-sum Test).

The "level" of one or more biomarkers, or constituents, means the absolute , relative or normalised amount or concentration of the biornarker or constituent in the sample. As used herein "control" or "control levei" indicates the level of a biornarker or constituent that is present in a sample without a particular condition, such as a healthy non-cancerous sample,, which may be a sampie without the prostate condition, or may be a sample with a benign prostate condition, such as benign prostatic hyperplasia. Such "levels" may be tailored to specific techniques that are used to measure levels of biomarkers, or constituents, in biological samples (e.g., !CP~MS, ICP-AES, EXDRF, colorimetrie detection, vo!tammetry etc.), where the levels of biomarkers or constituents may differ based on the specific technique that is used. The method may include measuring mass fraction levels of the constituents.

"Prostate cancer" refers to a disease in which cancer develops in the prostate, a gland in the male reproductive system,

"Benign prostatic hyperplasia" refers to a histologic diagnosi characterized by proliferation of the cellular elements of the prostate, a gland in the male reproductive system.

"Sampie" or "biological sample" means biological materia! isolated from a subject. The biological sampie may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non~ce!luiar materia! from the subject. The sampie can be isolated from any suitable biological tissue or bodily fluid such as, for example, prostate tissue, blood, blood plasma, urine, prostatic fluid, expressed prostatic secretion or seminal fluid.

"Subject" means any animai, but is preferably a mammal, such as, for example, a human.

"False positive" is a test result that indicates that a subject has a specific disease or condition when the subject actually doe no have the disease or condition.

"False negative" is a test result that indicates that a subject does not have a specific disease or condition when the subject actually has the disease or condition. "True positive" is a tes resuif thai indicates that a subject has a specific disease or condition when the subject actually has the disease or condition.

"True negative" is a test result that indicates that a subject does not have a specific disease or condition when the subject actually does not have the disease or condition. Test sensitivity is calculated using following equation:

Sensitivity = (True Positives (ΪΡ)/ίΤΡ + False Negatives {FN}]} X 100%

Test specific*/ is. calculated using foilowing equation:

Specificity ~ {True Negatives (TN)/[TN + False Positives {FP)j} X 100%

Test accuracy is calculated using following equation: Accuracy = [(TP+TN)/(TP+FP+TN+FN)] X 100%

"Combination" of levels of constituents refers to any mathematical relationship or manipulation between levels of two or more constituents. As described above, this may include a ratio between levels of constituents {or the quotient of constituents), such as Ca/Fe. It may also include a multiple (or product) of ratios, such as (Ca/Cu)*( g/Cu) ₍ or it may include a ratio {or quotient) between multiples (or products) of the levels of two or more constituents, such as {Ca ^* Mg*Zn)/(A!*Bi*Cu). The combination may also include the product of the levels of two or more constituents.

Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the foilowing description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BR!EF DESCRIPTION OF THE DRAWINGS

The appended drawings have been inciuded herein so that the cited features, advantages, and objects of the invention wilt become clear and can be understood in detail These .drawings form a pas of the ^■ specification. It is to he noted, however, that the appended drawings illustrate preferred embodiments of the inventio and should not be considered to limit the scope of the invention,

Fsgure 1 shows ^' individual data sets for A! mass fractions (mg/kg of dry tissue) in samples of normal {1 ), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 2 shows individual data sets for Ba mass fractions (mg/kg of dry tissue) ^' in sampies of norma! (1), benign hyperplastic (2) and cancerous prostate tissue (3), Figure 3 shows individual data sets for Bi mass fractions (mg/kg of d y tissue) in sampies of norma! (1 ), benign hyperplastic (2) and cancerous prostate tissue (3),

Figure 4 shows individua! data sets for Ca mass fractions (mg/kg of dry tissue) in samples of norma! (i), benign hyperplastic (2) and cancerous prostate tissue (3),

Figure § shows individual data sets for Mg mass fractions (mg/kg of dry tissue) in sampies of normal (1 ), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 6 shows individua! data sets for Mn mass fractions (mg/kg of dry tissue) in samples of norma! (1), benign hyperplastic (2) and cancerous prostate tissue (3),

Figur© 7 shows individual data sets for Ca/Fe mass fraction ratio in samples of ^■ normal (1 ), benign hyperplastic (2) and cancerous prostate tissue (3), Figure 8 shows individual data sets for Mg/AI mass fraction ratio in samples of norma! (1 ), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 9 shows individua! data sets for Ca/Cu mass fraction in sampies of normal (1 ), benign hyperplastic .(2) and cancerous prostate tissue (3).

Figure 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).

Figure 11 shows individua! data sets for {Ca/Gu)*(Zn/Cu) mass fraction ratios combination in samples, of norma! (1), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 12 shows individual data sets for < _. g/Cu) ^* (2n Cu) mass fraction ratios combination in sampies of normal (1 ), benign hyperplastic (2) and cancerous prostate tissue (3). Fsgure 13 shows individual data sets for Ca/Ba mass fraction ratio in sampies of norma! (1), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 14 shows individuai data sets for Ca/P mass fraction ratio in sampies of normal (1), benign h perp!asiic (2) and cancerous prostate tissue (3). Figure 15 shows Individuai data sets for Ga/Si mass fraction ratio in sampies of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3),

Figure 16 shows individual data sets for Ca/Sr mass fraction ratio in sampies of norma! (1), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 17 shows individuai data sets for Zn/Mn mass fraction ratio in samples of normal (1), benign hyperplastic (2) and cancerous prostate tissue (3),

Figure 18 shows individuai data sets for [(Zn ^* Ca ^* Mg*Cd)/(Si*Br*Ai*Fia) ^' j*lOOO mass fraction ratio in sampies of normal ( 1), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 19 shows individual data sets for

i(Ca d o ^* Rg ^* K ^* Mg ^* a ^* P¾ ^* S ^* Se ^* Z^^^

!*Sr)]*10 ¹⁸ in sampies of norma! (1 ), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 20 shows individuai data sets for

[(Ca d ^* Co1-ig ^* K ^* g ^* Na ^* P ^* Rb^

in samples of norma! (1 ), benign hyperplastic (2) and cancerous prostate tissue (3).

Figure 21 shows individual data sets for normalized mass fraction additive indices of four selected elements in sampies of normal, benign hyperplastic and cancerous EPS.

F gure 22 shows individuai data sets for normalized mass fraction multiplicative indices of four selected elements in samples of EPS from normal, benign hyperplastic and cancerous patients.

Figure 23 shows individual data sets for normalized mass fraction multiplicative indices of six selected elements in sampies of EPS from normal, benign hyperplastic and cancerous patients. Figure 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).

The following ©xampies are given for the purpose of fiiustrating the variou embodiments of the present invention and are not meant to limit the present invention in any fashion. SPECIFIC EXAMPLES

Example 1, Identification of c ncer blomarkers in prostate tissue using Inductively Coupled Plasma M ® Spectrometry {ICP~88S} and Induct vely Cou led Atomic Emission Spectrometry (ICP-AES),

Experimental conditions of the present study wer approximated, to the hospital conditions as closely as possible.

Equipment:

Autoclave (Ancon~AT2, Russia), inductively coupled plasma mass spectrometry instrument Thermo-Fisher "X-7" (Thermo Electron, USA), Spectrometer iCAP-61 (Thermo Jarre!! Ash, USA).

Specimen; 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. Reagents:

HNO (nitric acid 65 % for analysis, max. 0,005 ppm Hg, GR, ISO, Merck), H2O2 (hydrogen peroxide pure for analysis, Merck), ICP- S standards IGP- S-68A and ICP-Alv-6~A (High-Purity Standards, Charleston, SC 29423, USA), ICP stock solutions (High-Purity Standards, Charleston, SC 29423, USA). Protocol:

1.5 mL of H O3 and 0.3 ml- of H2O2 were added to homogenized and freeze-dried prostate tissue sample, placed in one-chamber autoclave, and decomposed for 3 hours at 160~2.Q0 ^P C, The heat- treated sample was cooled down to the room temperature; the soluble fraction was diluted with deionizeci water to 20 mL and transferred to a plastic measuring bottle. Simultaneously, the sam procedure was performed on a sample containing no prostate tissue, and the resultant solution was used as a blank sampie. Ail samples were analysed by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

The spectrometer parameters and the main parameters of ICR-MS measurements: generator output power 1 ,250VV, spray chamber cooled at 3°C, plasma gas flow rate - 12 L/min, nebuliser ~ Potycon, auxiliary air flow rate - 0.9 L/min, nebulizer flow rate - 0.9 Urn in, sampie update - 0.8 mL/min, resolution - 0.8, defector mode ~ double, scanning mode - survey scan (number of runs ■· 10, dwell time - 0.6 ms, channels per mass - 10, acquisition duration ~ 13.2 s) and peak jumping (sweeps -.26·, dwelt time— 10 ms, channels per mass - , acquisition duration - ^■■ 34 s).

The spectrometer parameters for !CP-AES measurements: generator output power 1 ,200 W, reflected power <5 W, nebuliser type - angular, plasma-forming air flow rate - 18 L/min, auxiliary air flow rate - 0.9 L min, air flow rate into atomiser -- 0.6 L/min, flow rate of the analysed sampie ~ 1.5 iTiL/min, zone height for plasma observation - 14 mm.

Results: The content of Ag, Ai, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, d, Ni, Pb, Pr, Rb, Sb, Sc, Se, Si, Sm, Th, Ti, U _s Y, and Zr in prostate tissue was analysed by ICP-MS. The content of Na, fvlg, P, S. K, Ca, Fe, Cu, n, Sr, and Ba in prostate tissue was analysed by ICP- AES.

Statistically significant differences in mass fraction levels of 45 chemicai elements (Table 1) were found in samples derived from cancerous, benign hyperplastic and norma! 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 1. Comparison of mean values (MtSEivi) of chemical element mass fractions (mg-kg- , dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) prostate tissue

Element Prostate tissue Ratios, p (t-tesf)

Norma! BPH PCa BPH PCa PCa 41-87 year 38-83 year 40-79 year to to to n=37 n=43 ^■ n=60 Normal Norma BPH

Ag 0.0284*0.0035 0.04G7±0.0Q88 0.255±0.031 1,43 8.98 ^c 6.27 ^c Ai 34.1+3.5 24.4+3.2 328±73 0.72 9.62 ^c 13.4 ^C

Au 0.0041 ±0.0008 0.QG26±G.0008 0.0297+0.0056 0.63 7.24* 11.4 ^C

B 1.04±0.18 1.51 ±0.26 12.6+3.7 1.45 12.1 ^b 8.34 ^b

Ba 1.48+0.21 1.22±0.20 26.7±7.6 0.82 18.0 ^b 21.9"

Bi 0,029±0.01 1 0.140±0.042 1.76±0.27 4.83 ^a 60.7 ^C 16.9 ^C

Br 27.9+2.9 30.0±2.6 99.9±8.9 1.08 3.58 ^c 3.33°

Ca 2397+235 2032± 85 675±S8 0.85 0.28° 0.33°

Cd 1.12+0.12 .07+0.43 0.425.+0.099 0.96 0.38 ^" 0.40

Ce Q.0309±0.0QS0 G.0128±0.0019 0.101±0.013 0.41 ^b 3.27° 7.89°

Co 0.0452±0.GG43 0.0716+0.0097 0.0326+0.0037 1.58 ^s 0,72 ^a Q.46 ^b

Cr G.53±0.Q8 1 .07+0.12 2.35+0,37 2.02 4.43 ^c 2.20 ^b

Cs Q.0339±0.QQ33 0.0235+0.0025 0.0389+0,0039 0.69 ³ 1.14 1.86 ^b

Cu 9.85+0.97 9.86±1.25 17.1 +2.0 1 ,00 1.74 ^b 1,74 ^b

Dy 0.0029±0.0005 0.0016±G.0002 0.0072±0.0011 0.53 ^s 2.48 ^c 4,50 ^c

Er 0.00148±0.0002 0.00072±0.0001 0.00297±0.0003

3 3 8 0.49 ^b 2.01 4,13 ^c

Fe 1 1 ±9 139±10 165+15 1.25 1.49 ^a 1.19

Gd 0.0029±0004 0.0015±0.0003 0.0094±0.001 0.52 ^b 3.24 ^e 6.27 ^c

Hg G.052±0.0Q9 0.275±0.036 Q.13Q±Q.021 5.29 ^c 2,50 ^c 0.47 ^b

Ho 0.00057±0.0000 0.00032+0.0000 0.001 8±0.0002

8 δ 2 0.56 ^a 3.12° 5.56 ^c

12030±475 14472±740 8542±504 1.20 ^b 0.71 ^c 0.59°

La 0.080±0.019 0.019±0.003 0.970±0.540 0.24 ^b 12.1 51.1

Li 0.0419±0.0055 O.0385±0.0073 0.251±0.054 0.92 5,99 ^b 6.52 ^a

Mg 071 ±76 1201 ±83 346±61 1.12 0.32: 0.29 ^c

Mn 1 ,32+0. OS 1.19±0.09 6.99±1.35 0.90 5.30 ^c 5.87 ^c

Wa 10987±394 11612+869 7511 ±843 1.08 0.88 ^s 0.65 ^c

Nd 0.0 37±0.0021 0.0062+0.0009 0.0413+0.0065 0,45 ^b 3.01 ^c 6.68 ^c Ni 3.10+0,51 3.22±1.06 6.96+1.04 1.04 2.25 ^c 2,16 ^b

P 7817+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 ^a 0.76 2.62 ^b

Pr 0,0035±0.0005 0.0015+0.0003 0.0097±Q,0017 0.43 ^b 2.77 ^b 6.4.7°

R 14.8+0.9 14.4±Q.7 8.8±0.7 0.97 0.59° 0.61 ^c

S 8557±254 8787+487 5343+389 1.03 0.62 ^c 0.61 ^c

Sb 0.037*0.005 Q.142±0.036 0 ^' .501±0.062 3.84 ^b 13.5 ^C 3.53

Sc 0.0294+0,0053 0.0257*0.0040 0.0116±0.0015 0.87 0.39 ^s 0.45 ^b

Se 0.696±0.044 1.243±0.079 0.676±0.078 1.79 ^c 0.83 0.46 ^c

Si 102+11 141 ±24 284±39 1.38 2 8 ^C 2.02 ^b

Sm 0.0027+0.0004 0.0014.+0.0004 0.0095+0.0029 0.52 ^B 3.52 ^a 6.71 ^b

Sr 2.34+0.38 3.69±0.45 5.75+0.60 1.58 ^s 2.46° 1.56 ³

Th 0.0034+0.0007 0.0018+0,0003 0.0490+0.0120 0.52 ³ 14.4 ^C 27,2 ^C

T! 0.0014+0.0002 0.0020+0.0006 0.0219+0.0056 1.43 15,6 ^C 1 1.0 ^b

U 0.0070+0.0021 0.0021 +0.0009 0.0068+0.0013 0.30 ³ 0.97 3.24 ^h

Y 0.0'S86±0.0042 0.0071 ±0.0012 0.0340+0.0038 0,38 ^a 1.83 ^b 4.79=

Zn 1061+153 1136+96 136±9.9 1.07 0.13 ^C 0.12 ^C

Zr 0.036±0.006 0.091+0.036 2.13+0.89 2,53 59.2 ^a 23.4 ^s - arithmetic mean, SEM - standard error of mean, ^a ~ p±0.05, ^b - ,o+0.0i , ^c - p±Q.00i.

Example 2. Establishing the prostate condition using AI mass fraction in prostate tissue sample. The tissue content of Ai was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Example 1 , Tabie 1 ). Mass fraction of Ai in tissue of normal prostate was found to be 34.1 ±3.5 (SEM) mg/kg, in BPH 24.4+3.2 (SEM) mg/kg, and in PCa 328±73 (SEM) mg/kg on dry mass basis (Table 1 ). The upper limit for AI mass fraction in dry norma! and BPH prostate tissue was determined to be M+25D or 70 mg/kg on dry mass basis (Figure 1 ). if PCa needs to be discriminated from norma! and BPH tissue and if Ai content in a prostate biopsy sample prepared and analyzed as described in the Example t exceeds 70 mg kg dry tissue, prostate carcinoma with an accuracy of 82+1.2% can be diagnosed. The sensitivity and specificity of the Ai based test is 97±3% and 94±4%, respectively, Example 3. Establishing the prostate condition using Ba mass fraction in prostate tissue sample.

The tissue content of Ba was found to be significantly different in most cancerous prostate tissues as compared to normal and benig hyperplastic tissues (Example 1, Table 1 ). Mass fraction of Ba in tissue of normai prostate was found to be 1.48±0.21 (SEM) mg/kg, in BPH 1.22+0.20 (SEM) mg/kg, and i PCa 26.7±7.8 (SEM) mg/kg on dry mass basis (Table 1 ). The upper limit for Ba mass fraction in dry normal and SPH prostate tissue was determined to be M+2SD or 3.5 mg/kg on dry mass basis (Figure 2). if PCa needs to be discriminated from norma! 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 a accuracy of 82+1 % can be diagnosed. The sensitivity and specificit of the Ba based test is 97±3% and 94±4%, respectively.

Example 4, Establishing the prostate condition using 81 mass fraction in prostate tissue sample.

The tissue content of Bi was found to be significantly different in most cancerous prostate tissues as compared to norma! and benign hyperplastic tissues (Example , Table 1 }. Mass fraction of Bi in tissue of normal prostate was found to be 0.029+0.0 1 (SEM) mg/kg, in BPH 0.140±0.042 (SEM) mg/kg, and in PCa 1.78±0.27 (SEM) mg/kg on dry mass basis (Table 1 ). The upper limit for Bi mass fraction in dry normai and BPH prostate tissue was determined to be M+2SD or 0.5 mg/kg on dry mass basis (Figure 3). If PCa needs to be discriminated from normai and BPH tissue and if Bi content i 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 82+12% can be diagnosed. The sensitivity and specificity of the Bi based test is 97±3% and 93±4 , respectively.

Example 5, Establishing the prostate condition using Ca mas fraction in prostate tissue sample.

The tissue content of Ca was found to be signif icantly different in most cancerou prostate tissues as compared to norma! and benign hyperplastic tissues (Example , Table 1). Mass fraction of Ca in tissue of normal prostate was found to be 2397±235 (SEM) mg/kg, in BPH 2032±165 (SEM) mg kg, and in PCa 875±58 (SEM) mg kg on dry mass basis (Table 1 ). The upper limit for Ca mass fractiori in dry cancerous prostate tissue was determined to be M+2SD or 1080 mg/kg on dry mass basis (Figure 4), if PCa needs to be discriminated from norma! and BPH tissue and if Ca content in a prostate biopsy sample prepared arid 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 sensitivit and specificity of the Ca based test is 98% and 97%, respectively. Exam le 0. Establishing the prostate condit on using Wg mass fraction in prostate tissue sample,

The tissue content of Mg was found to be significantly different in most cancerous prostate tissues a compared to normal and benign hyperp!asfic tissues (Example 1 , Table 1 ), ass fraction of Mg in tissue of normal prostate was found to be 1071±7 (SEM) mg/kg, in BPH 1201 ±83 (SEM) mg/kg, and in PCa 346±61 (SEM) mg kg on dry mass basis (Table 1 ). The upper limit for Mg mass fractio in dry cancerous prostate tissue was determined to be M+2SD or 700 mg/kg on dry mass basis (Figure 5). if PCa needs to be discriminated from normal and BPH tissue and if Mg content in a prostate biopsy samp!e prepared and analysed as described in the Example 1 does not exceed 700 mg/kg dry tissue, prostate carcinoma with an accuracy of 90±4% can be diagnosed. The sensitivity and specificity of the Mg based test is 100-10% and 84±6 %, respectively.

Example 7» Establishing the prostate condition using IVIn mass fraction in prostate tissue sample.

The tissue content of Mn was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues (Exampie 1, Table 1). Mass fraction of Mn in tissue of normal prostate was found to be 1.32+0.08 (SEM) mg/kg, in BPH 1.19±0>09 (SEM) mg/kg, and in PCa 6.99±1.35 (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 IVH-2SD or 2 mg/kg on dry mass basis (Figure 6). 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 Exampie 1 exceeds 2 mg/kg dry tissue, prostate carcinoma with an accuracy of 96±3 can be diagnosed. The sensitivity and specificity of the Mn based test is 91. ±9% and 97+3%, respectiveiy,

Example 8. Determination of mass fraction levels of 44 elements relative to the mass fraction of ^' calcium in normal, cancerous and BPH prostate tissue.

Mass fraction ratios of the elements mentioned in the Example 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 fo 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 2, Means of ratios (Iv!lSEM), their ratios and the reliability of difference between mean values of mass fraction ratios of Ca to mass fractions of other chemical element in normai, benign hyperplastic (BPH) and cancerou (PCa) prostate tissue.

Mass Prostate tissue Ratios of means, p (t~tesi) fraction

ratio Norma! BPH PCa BPH PCa PCa

41-79 year 38-83 year 40-79 year to to to n=*37 n=43 n=60 Normai Normai BPH

Ca/Ag 0703 ± 15763 1 7550+34515 4164+1611 1 ,10 0,039 ^c 0,036 ^b

Ca/AI 103±21 101+18 5.24+1.9 0,98 0,051 ^c 0,Q52 ^C

Ca/B 4320±80S 1550+191 119±58 0,36 ^b 0,028 ^Γ - 0,077°

Ca Ba 2957±577 2034±251 102±47 0,050

0,69 0,035° c

Ca/Bi 532698+114578 93934*41193 7501 ±6 39 0,18 ^C 0,014 ^C 0,080 ^»

Ca Br 91.3.+12.7 78.9±14.6 11.1+4. 0,86 0, 2 ^C .0,14°

Ca Cd 3085±455 3753±732 2002+212 1 ,22 0,65 ^a 0,53 ^a

Ca Ce 144087+28909 19 735.+31 86 8862±2222 1 ,33 0,062 ^c 0,046 ^c

Ca/Go .69329*11034 42314+4982 13669*1072 0,61 ^a 0,20° 0,32°

Ca Cr 145 6±6572 2169±218 191+41 0,15 0,013 ^a 0,088 ^c

Ca/Cs 94882+17335 92843+9485 22723+6474 0,98 0,24 ^c 0,24 ^c Ca/ ^' Cu 315+47 223±23 56+14 0,71 0,18 ^C 0,25 ^c

Ca/Dy 161389+4788

1733952+444593 1685620+327920 9 0,97 0,083° 0,096°

Ca/Er 296687±6492

2782727±557202 3989832±8451 9 4 1 ,43 0,11° 0,074°

Ca/Fe 20.8±2,4 17.8 1.9 4.81 ±0.53 0,86 0,28° 0,27°

Ca/Gd 118454+3567

1489869±334486 1726552±327832 7 1 ,16 0,080° 0,069°

Ca Hg 78 86+11882 9944+1259 4445±2330 0,13° 0,057° 0,45 ^a

Ca/Ho 4S3653+8828

7482S30±1547085 8358623+1644445 4 1 ,12 0,061 ^c 0,054°

Ca/K 0.227±0.037 0.144+0.013 0.081+0.008 0,63 ^a 0,38° 0,56°

Ca/La 105705±19452 128328±16885 7340+3488 1 ,21 0,069° 0,057°

Ca Li 79700+10834 71782+11904 5847+2025 0,90 0,073° 0,082°

Ca/Mg 2.83±0.51 1.72+0.12 2.58+0.47 0,81 0,91 1 ,50

Ca/Mn 2061 ±325 1789±188 181+66 0,87 0,088° 0,10°

Ca/Na 0.236±O.G32 0.189+0.025 0.097±0.014 0,80 0,41° 0,51 ^b

Ca/Ni ^' 1916x626 1028±179 23±28 0,54 0.064» 0,12°

Ca/P 0.348+Q.040 0.284+0.025 0.112*0.020 0,76 0,32° 0,42°

^■ Ca/P 3774+724 44811756 558+149 1 ,18 0,15° 0,12°

Ca/Pr 112525±3521

1263853+289288 2231480+735010 8 1 ,77 0,089? 0,050*

Ca/ b 180+23 139+12 81.8+7.8 0,77 0,45° 0 59·

Ca S 0.308+0.045 0.238±0.022 0.133+0.016 0,77 0,43 ^c 0,56°

Ca/Sb 121963+24741 49028+20319 2784+556 0,40 ^s 0,023° 0,057 ^s

Ca/Sc 1 49S8±51707 76930±10995 49945+6858 0,44 0,29a 0,65 ^a

Ca/Se 3804±500 2362±296 895±168 o.ee* 0,25° 0,38°

Ca/'Si 35.7±6.9 17.4+2.3 3.21+0.88 0,49* 0,090° 0,18°

Ca/Sra 1695658±438262 2939100+810027 1 ,73 0,084 ^b 0,048°

142073±3759 6

Ca/Sr 1334±142 743±76 137+34 Q,56 ^b 0,10° 0,18 ^C

Ca/Th 1703628±375869 1499284±275745 43707±21300 0,88 0.026 ^c 0,029'

Ca/T! 124174±7490

2870589±627543 14641 3±252751 3 0,51 ³ 0,043= 0,085=

Ca/U 130793±22Q7

1122953+182815 2061625*434930 3 1 ,84 0,12* 0,063=

Ca Y 414438*116105 385834±74931 23034+3883 0,93 0,056 ^s 0,060=

Ca/Zn 3,89±0.91 1.72±0.21 5.02+0.41 0.44 ^a 1.29 2.92 ^f; /Z 135701 ±31300 61766+18949 853+238 0,46 ^a 0,0063= 0,014=

M - arithmetic mean, SEM - standard error of mean, ⁸ - p≤0.05, ^¾ - p<0.0 , ^c ~ p≤Q,G01.

Example 9. Ostermln alien of mass fraction levels of 44 elements relative to mass fraction of zinc in normal, cancerous and BPH prostate tissue.

Mass fraction ratios of the elements mentioned in the Example 1 are different i 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 norma! 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 3. Means of ratios ( +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

Mass Prostate tissue Ratios, p (Hesi) fraction ___ __

ratio Normal BPH PCa PCa ^~

41 -79 year 38-83 year 40-79 year to to to n=37 n=43 n~60 Normal Nonnal BPH

Zrt/Ag 32271.+5360 39748±4328 723±133 1.23 0.022= 0.018=

Zn/AI 41.3+9.7 59.0+9.8 1.16±0,52 1.43 0.028= 0.020= Zn/Au 645790+240530 816590±1 36 0 19120112210 1.27 Q.029 ^b 0.023°

Zn/B 1974+559 360±417 28.8±21.0 0.69 0.015 ^b 0.021 °

Zn/Ba 003+195 1373+203 30± 7 1 .37 0.030° 0.022°

Zn/Bi 236160+82300 79960+37890 2290+2110 0.34 ^a 0.0097° 0,029*

Zn/Br 39.1 +6.2 88.8+11.5 1.30+0.14 1.76 ^s 0.033° 0.019°

Zn Ca 0.449+0.059 0.758±0.171 0.169+0.027 1.69 0.38 ^f; 0.22 ^b

Zn Cd 39 70+1 900 39 Q±6460 319±59 1.00 0.0082° 0.0082'

Zn/Ce 60330+14060 131210+22300 2055±899 2.17 ^b 0:035° 0.016°

Zn/Co 27011+3716 20798+3359 4293+554 0.77 0.16 ^C 0.21°

Zn/Cr 2654+356 1 61+156 78.1 ±13.4 0.44° 0.029° 0.067°

ZrtfCs 37990±S990 69G50±15160 3899+ 58 1.82 0.103' ^· 0.057°

Zn/Cu 114±19 133±19 9.0+2.3 1.17 0.079° 0.068°

Zn Dy 1 94200+25554

657590+148330 0 30310±10770 1.81 0.046° 0.025°

Zn/Er 1190700±29366 2796660+59075

0 0 54040+15720 2.35 ^s 0.045° 0.019°

Zn/Fe 8.8±1.4 1 .8+1.5 0.97+0.1 1.34 0.11° 0.082°

Zn/Gd 1190620+24026

624740±158040 0 23210±8250 1.91 0.037° 0.019°

Zn/Hg 27011 ±3717 6490+688 1216+115 0.24° 0.045° 0.19°

Zn Ho 3128380± 76622 5591120+95864

0 0 973 Q±37950 1.79 0.031° 0.017°

Zn/ 0.G86.+0.016 0.109±0.024 0.0135±0.002

6 1.27 0.16° 0.12°

Zn/La 6 550+25120 96668±23120 2 56+1250 1.57 0.036* 0.022°

Zrv'Li 35526+9597 51562+11566 1248+523 1.45 0.035 ^b 0,024°

Zn/Mg 1.01 ±0. 4 .33±0.35 0.38+0.50 1,32 0.38 0.29

Zn/Mrr 847+.21Q 1261 ±185 43±23 1.49 0.051° 0.034°

Zn/Na 0.099+0.01 0.144+0.035 0.0153±0.002 .45 0.15° 0.11 ^b Zn/ d 133860±32890 275460±44Θ60 4690*1810 2,05 ^a 0.035 ^c 0,017=

2n Ni 712*185 820+220 26*11 1.15 0.037= 0.032=

Zn/P 0.128*0.016 Q.198±0.041 0.0187*0.004

2 1 .55 0.15 ^G 0.094 ^c

Zri/Pb 1523±348 291 *470 128*58 1.91 ³ 0.084° 0.044°

Zn/Pr 1429530*34874

529125± 130900 0 21630*7700 2.70 ^a 0.041= 0,015°

Zn/Rb 71.7*9,0 87.4±9.3 17.9*2.0 1.22 0,26= 0,22=

Zn/S 0.123*0.025 0.182*0.041 0.0213*0.003

5 1.48 0.17° 0,12°

2n/Sb 34333*6156 10 15*2344 334*44 0.29° 0.0097= ^■ 0.033°

Zn/Sc 46794*7866 39678*3372 13157*1624 0.85 0.28° 0.33°

Zn/Se 1548*166 886*90 270*28 0.57° 0.18= 0.30=

Zn/Si 15,8±4.9 14,3*4.6 0.69*0.33 0.91 0.044= Q.048 ^b

Zn Sm 1796120*41320

642630*151110 0 27835*9730 2J9 ³ 0.043 ^c 0.015=

Zn/Sr 56 ±83 641*223 22.2*4.7 1.14 0.040= 0.035 ^fe

Zn/Th 1041050*21934

721 80*196050 0 12010*7530 1.44 0.017° 0.012=

Zn/TS ^■ 855140±116010 877340*132760 35010*26220 1.03 0,041= 0.040=

Zn/U 1514290*37828

461270+98980 0 23375*6170 3.28 ^b 0.050= 0.015=

Zn/Y 171540*52820 271240*54800 4540*1495 1 .58 0,026 ^b 0.017=

Zn/Zr 49 00*9570 41930* 1960 151*58 0.85 0.0031= 0.0036=

M ~ arithmetic mean, SE ~ standard error of mean, ³ - p≤0.05, = p<0.001.

Example 10. Usmg the Ca/Fe mass fraction ratio to establish son.

The Ca/Fe mass fraction ratio was found to be significantl different in most cancerous prostate tissues as compared to norma! and benign hyperplastic tissues (Exampie 8, Tabie 2). The upper limit for Ca/Fe mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (M - arithmetic mean, SD - standard deviation) or 10 (Figure 7), if PCa needs to be discriminated from normai and BPH tissue and if the Ca/Fe ratio in a prostate biopsy sam ie prepared and analysed as describee! in Example 1 does not exceed 10, prostate carcinoma with an accuracy of 98±3% can be diagnosed. The sensitivity and specificity of the Ca/Fe ratio based test is 100-9% and 95±4%, respectively.

Example 11, Using the g/AI mass fraction rati© to establish prostate condition.

The Mg/AI mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to norma! and benign hyperplastic tissues. The upper limit for Mg/A! mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+2SD ( - arithmetic mean, SD - standard deviation) or 9 (Figure 8). if PCa needs to be discriminated from normal and BPH tissue and if the Mg/AI ratio in a prostate biopsy sample prepared and analysed as described in Exampie 1 does not exceed 9, prostate carcinoma with an accuracy of 99% can be diagnosed. The sensitivity and specificity of the Mg/A! ratio based test is 98% and 99%, respectively. Example 12, Using the Ca Cu mass fraction ratio to establish prostate condition.

The Ca/Cu mass fraction ratio was found to be significantly different In most cancerous prostate iissues as compared to norma! and benign hyperplastic tissues (Exampie 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 (M - arithmetic mean, SD - standard deviation) or 100 (Figure 9). if PCa need to be discriminated from norma! 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 00, prostate carcinoma with an accuracy of 97±3% can be diagnosed. The sensitivity and specificity of the Ca Cu ratio based test is 91 ±9% and 100-3%, respectively.

Example 13. Using the (Ga Gu)*{ g/Gu) mass fraction ratio combination to establish prostate condition,

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 iissues. The upper limit for (Ca/Cu)*(IV1g/Cu) mass fraction ratio combination on dry mass basis in cancerous prostate tissue was determined to be +3SD ( - arithmetic mean, SD - standard deviation) or 4000 (Figure 10). 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)*(ivg/Cu) ratio based test is 100-11% and 100-3%, respectively. Example 14. Using the (€a/Cu) ^* (Zn/Cu) mass fraction ratio combination to establish prostate condition,

The (Ca/Cu) ^* (Zn/Cu) mass fraction ratio combination was found to be significantl different in most cancerous prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for (Ga/Cu)*{Zn/Cu) mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be M+3SD (M - arithmetic mean, SO - standard deviation) or 1700 (Figure 11 ).

If PCa needs to be discriminated from normal and BPH tissue and it ^" the (Ca/C ^' u) ^* (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.

The {Mg/Cu) ^* (Zn/Cu) mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to norma! 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 ( - arithmetic mean, SD - standard deviation) or 975 (Figure 12). 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 (iv!g/Cu)*(Zn/Cu) ratio based test is 100-11 % and 100-3%, respectively. Example 18. Using bodily fluids and tissues to establish prostate condition.

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-vaiue) 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 eiacuiate and is present in urine too; therefore the concentration of the specific biomarkers will also be reflected in ejaculate and urine. As result, prostate condition can be established using the biomarkers given in the Table 1 using prostatic fluid, seminai fluid and urine samples.

Table 4. Correlation coefficient (r~value) between the mass fraction of Ca arid Mg in prostate tissue and the relative voiume of the main histological compartments of prostate tissue.

CornpartfTien /Eiement Ca Mg

Glandular epithelium 6Vi 4 0.385

Stroma -0.421 -0.482

Lumen 0 _S 582 0.437

Statistically significant r-vaiues are given in bold

Example 17. Identification of cancer biomarkes in expressed prostatic secretion using Inductively Coupled Plasma ass Spectrometry (ICF-MS) a d Inductively Coupled Atorolc Emission Spectrometry f CP-AES). Experimental conditions of the present study were approximated to the hospital conditions as closely as possible.

Equipment: inductiveiy coupled plasma mass spectrometry instrument Agilent 7500c. Specimen: 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), TransRectai Ultrasound imaging (TRUSI) and microscopic analysis of tissue morpholog in biopsies obtained from the same patients, The absence of cancer was confirmed by DRE and TRUSI. Reagents:

HNO3 (nitric acid 85 % for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H2O2 (hydrogen peroxide pure for analysis, Merck), ICP-MS standards NCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureau of Reference of the European Gomlssion) and IRM- BD151 (LGC Standards, Weisef, Germany),

Protocol:

0,5 ml., of HNOa was added to freesre-dried EPS sampies and the samples were left over night at room temperature. After that 0.25 mL of H O3 and 0.15 ml of H2O2 were added to the samples and placed in water bath at 95°C for 30 min. The heat-treated sampies were eooied down to the room temperature; the soluble fraction was diluted with detonized 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 soiution was used as a blank sample. Aii samples were analysed by Inductively Coupled Plasma Mass Spectrometry and inductively Coupled Plasma Atomic Emission S ectrometry.

The spectrometer parameters and the main parameters of ICP-SV1S measurements: auxiliary air flow rate - 0.9 L/min, nebulizer fiow rate - 0.9 L/min, sample update— 0.8 mLJmtrs. The spectrometer parameters for iCP-AES measurements: generator output power 1 ,500 W,

Results:

The content of Ag, A!, 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, T!, U, Y, and Zr in EPS was analysed by ICP-MS. The content of a, Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in EPS was analysed by IGP-AES.

Statistically significant differences in mass fraction levels of 46 chemical elements (Table 5) were found in sampies derived from cancerous, benign hyperplastic and norma! prostate EPS. Differences in mass fraction ievels 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 S. Comparison of mean values of chemicai element mass fractions (mg- kg ^{' !} , dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) EPS.

Prostatic Ratios of

fluid means

BPH to PCa to PCa to

Element Normal BPH PCa

Normal Norma! BPH

Li 0.45 0.18 0.21 0.4 0.5 Ϊ2 ^' 8 0.97 2.54 1,00 2.6 1.0 0.4

Ma 45440 47804 167000 1.1 3.7 3.5 g 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 1 040 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

Mrs 1.10 0.71 3.95 0.7 3,9 5.6

Fe 5.7 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

R 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 8.3

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

Ls 0.06 0.02 0.04 0,4 0.8 2.3

Ce 0.01 0.02 0.08 1.2 8.2 5.1

Pr 0.06 0.01 0.02 0.2 0.4 2.0

Nc! 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

Gci 0,01 0.01 Q.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

Ti 0.01 0.01 0.15 0.9 13.8 15.3

Pb 0.08 0.32 1.03 3. 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 .8 U 0.01 0.01 0.05 1.0 4.8 .8

Example 18. Identification of cancer bsofnarkers in seminal fluid using inductively Coupled Plasma Mass Spectrometry (ICP»MS) and Inductively Coupied Atomic Emission Spectrometry (ICP-AES). Experimental conditions of the present study were approximated to the hospital conditions as closely as possible.

Equipment; inductiveiy coupled plasma mass spectrometry instrument Agilent 7500c. Specimen: 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 TRUSS. Reagents:

HNOs (nitric acid 65 % for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H2O2 (hydrogen peroxide pure for analysis, Merck), ICR-MS standards NCSZC 73013 (NCS Certified Reference Material), 8C 063R (Community Bureau of Reference of the European Comission) and iRM- BD151 (LGC Standards, vVeise!, Germany),. Protocol:

0.5 mL of HNO3 was added to freeze-dried seminal fluid samples and the sam les were left over night at room temperature. After that 0.25 mL of HNOs and 0.15 mL of H2Q2 were added to the samples and placed in water bath at 95°C for 30 rnin. The heat-treated sample wa 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 Coupied Plasma Mass Spectrometry and Inductiveiy Coupled Plasma Atomic Emission Spectrometry. The spectrometer parameters and the main parameters of iCP-MS measurements; auxiliary air flow rate - 0.9 L/min, nebulizer flow rate ^■■■■ 0.9 L/min, sample update - 0.8 mL/min. The spectrometer parameters for ICP-AES measurements: generator output power 1 ,500 W.

Results: The content of Ag, AI, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd, Hg, Ho,- La, Li, Mn, Nd, , Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sra, Tb, Th, Ti, 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.

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 nom a! subjects. Differences in mass fraction levels of thes 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 8. Comparison of mean values of chemicai element mass fractions (mg- kg ¹ , dry mass basis) in normal, benign hyperplastic (BPH) and cancerous (PCa) seminal fluid.

Seminal i Ratios of

fluid I means

BPH to PCa to PCa to

Element Nonna! BPH PCa

Normal Norma! 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 1 100 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 18 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 13 2.91 2.2

Mn 0.23 0.35 0.12 1.6 0.55 0.4

Fe 19.0 7.3 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 2.67 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 0.1 0.01 1.0 0.10 0.1

Cs 0.01 0.06 0.01 4.5 0.99 0.2

8 a 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 d 0.01 0.0 0.01 1.0 1.00 1.0

Sm 0.01 0.01 0.0Q 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 0.0? 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

Tl 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

Exam le 19. Establishing the prostate condition using Mn mass fraction In an EPS sample.

The tissue content of Mn was found to be significantly different in most cancerous EPS samples as compared to normai and benign hyperplastic EPS sampies (Example 17, Tabie 5). Mass fraction of Mn in EPS of normai 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 normai or BPH subject was determined to be M+2SD or 2.4 mg/kg on dry mass basis. if PCa EPS needs to be discriminated from normai and BPH and if Mn content in the EPS sample prepared and analysed a described in the Example 17 exceeds 2.4 mg/kg dry EPS, prostate carcinoma can be diagnosed with an accuracy of 94±3%. Exa pSe 20, Establishing the prostate condition using AI m ss fraction in an EPS sample.

The tissue content of A! was found to be significantly different in most cancerous EPS samples as compared ίο· normal and benign hyperplastic EPS samples (Example 17, Table 5). Mass fraction of A! in EPS of norma! 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 AI mass fraction in dry EPS from norma! or BPH subject was determined to foe +2SD or 25 mg kg on dry mass basis. if EPS PCa needs to be discriminated from norma! and BPH and if AI 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 98±4% can be diagnosed. Example 21 , Establishing the prostate condition using Ba mass fraction in an EPS sample.

The tissue content of Ba was found to be significantly different in most cancerou prostatic fiuid samples as compared to norma! 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 (Tab!e 5). The upper limit for Ba mass fraction in dry EPS from norma! or BPH subject was determined to be M+2SD or 1.5 mg kg on dry mass basis.

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 1 exceeds 2.5 mg/kg dry EPS, prostate carcinoma with an accuracy of 95±5% can be diagnosed. Example 22. Establishing the prostate condition using Bl mass fraction in an EPS sample.

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, arid in PCa 0.40 mg/kg on dry mass basis (Table 5). The uppe 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. if EPS PCa needs to be discriminated from no iai 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 96±3% can be diagnosed.

Example 23, Establishing the prostate condition using Ca mass fraction in an EPS sample. The tissue content of Ba was found to be significantly different in most cancerous prostatic f!uid samples as compared to norma! and benign hyperplastic prostatic fiuid samples (Example 17, Table 5). Mass fraction of Ca in EPS of norma! 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 iower limit for Ca mass fraction in dry EPS from norma! or BPH subject was determined to be 8000 mg/kg on dry mass basts.

If EPS PCa needs to be discriminated from norma! and BPH and if Ca content in the EPS sample prepared and analyzed as described in the Example 1 does not exceed 2000 mg/kg dry EPS, prostate carcinom with an accuracy of 95±5% can be diagnosed.

Example 24. Establishing th© prostate condition using Mg mass fraction in an BPS sample. The tissue content of Mg was found to be significantly different in most cancerous prostatic fluid samples as compared to norma! and benign hyperplastic prostatic fluid samples (Example 1.7, Table 5). Mass fraction of Mg in EPS of norma! 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 Iower limit for Mg mass fraction in dry EPS from normal or BPH subject was determined to be 3500 mg/kg on dry mass basis. If EPS PCa needs to be discriminated from norma! and BPH and if mg content in the EPS sample prepared and analysed as described in the Example 7 does not exceed 3500 mg/kg dry EPS, prostate carcinoma with an accuracy of 9S±5% can be diagnosed.

Example 25. Establishing the prostate condition using Cr mass fraction In a seminal fluid sample. The tissue content of Cr was found to be significantly different in most cancerous seminal fiuid samples as compared to normal and benign hyperplastic seminal fluid samples (Example 18, Table 6). Mass fraction of Cr in seminal fluid of nom-iai 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. 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 94±3% can be diagnosed.

Example 26. Establishing the prostate condition using fVfg mass fraction m a seminal fiuid sample. The tissue content of Mg was found to be significantly different in most cancerous seminal fluid samples as compared to norma! and benign hyperplastic seminal fluid samples (Examp!e 18, Tab!e 6). Mass fraction of Mg in seminal fluid of norma! prostate was found to be 1 100 mg/kg, in BPH 3874 mg/kg, and In PCa 140 mg/kg on dry mass basis (Tabie 6). The lower limit for Mg mass fraction in seminal fluid from norms! or BPH subject was determined to be 700 mg/kg on dry mass basis. if PCa needs to be discriminated from norma! 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 92±5% can be diagnosed.

Example 2@. Establishing the prostate condition using Ca mass fraction i a seminal fluid sample, 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 2.200 mg/kg on dry mass basis.

If PCa needs to be discriminated from norma! 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 90±5% can be diagnosed.

Example 27, Determination of mass fraction levels of 44 elements relative to the mass fraction of calcium in normal, cancsrous and BPH EPS,

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 Tabie 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 ?. 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. Prostatic Ratios of

fluid means

BPH to PCa to PCa to

Element Normal 8PH 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/AS 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 8189 1 976 160 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 32711 236917 44829 0.7 0.1 0.2

Ca/Ni 34350 2350 541 0.1 0.02 0.2

Ca/Cu 304 788 263 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 41 9 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 3 319 30715 1148 1.0 0.04 0.04

Ca/La 199512 549579 84542 2.8 0.4 0.2

Ca/Ce 8593 1 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 1 04000 1019233 156230 0.9 0.1 0.2

Ca/Gd 1104000 903780 106736 0.8 0.1 0.1

Ca/Tb 1 104000 1075800 263046 1.0 0. 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 1 04000 1075800 151557 1.0 0.1 0.1

Ca/Au 54190 57363 380000 0.4 2.5 6.8

Ca/Cd 431048 126876 10313 0.3 0.02 0.1

Ca/Hg 143862 215160 76000000 1.5 528.3 353>2 ~3S~

Ca/T! 995043 1075800 24762 1 , 1 0.02 0.02

Ca/Pb 130574 33968 3684 0.3 0.03 0.1

Ca/Bi 671533 1066778 9446 1.6 0.01 0.0

Ca/Th 431082 926283 86428 2.1 0.2 0.1

Ca/U 1 104000 1075800 79838 1.0 0.1 0.1

Example 27. Determination of mass fraction levels of 44 elements relative i& the mass fraction of zinc in normal, cancerous and BPH EPS,

Mass fraction ratios of the elements mentioned in the Example 1 are different in non-cancerous and cancerous EPS and therefore these can be used as prostate tumor biornarkers. in the Tab!e 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.

Tafefe 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, be ign hyperplastic (BPH) and cancerous (PCa) subjects,

Mass Prostatic I Ratios of fraction fiuid means

BPH to PCa to PCa to ratio Norma! BPH PCa

Normal Normal BPH

Zn/Li 19121 28686 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

2n/A! 1880.0 407.9 4S.6 0.22 0.02 0.1 1

Zn/Si 333.6 1 15.0 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 0.88 1. 1

Zn/Sc 150218 92808 200000 0.62 1.33 2.15

Zn/Cr 14179 5678 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 1 12292 23594 0.44 0.09 0.21

Zn Ni 28777 1 1 14 285 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 Ζπ/Br 270.3 1 7.2 3.6 0.43 0.01 0.03

Zn/ b 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 331 66 13652 2129 0.04 0.01 0. 6

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 167 0.29 0.17

Zn/Ce 669858 331642 25287 0.50 0.04 0.08

Zn Pr 156473 476691 91590 3.05 0.S9 0.19

Zn/Nd 772531 184524 48182 0.24 0.06 0,26

Zn/StT! 860600 483089 32226 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 0.1 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

Zrs/Hg 2145 101980 40000000 0.91 356.68 392.23

Ζπ/ΊΊ 775665 509900 13033 0.66 0.02 0.03

Zn/Pb 101 86 16100 1939 0.16 0.02 0.12

Zn/Bi 523479 500884 4972 0.96 0.01 0.01

ZiVTh 336041 439033 45488 1.31 0.14 0.10

Zn/U 860600 509900 42020 0.59 0.05 0.08 i ple 27. D@t&rtnijiaiiori of mass fras ϊίίοη levels of 4 4 elements ι rslailve to th © mass fraction of calcium in seminal fluid from normal, cancerous and BPH subjects.

Mass fraction ratios of the elements mentioned in the Example 8 are different in non-cancerous and cancerous seminai 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 seminai fluid from normal, BPH and cancerous subjects are given. The data in the Table 9 aiiow evaluating the importance of individual mass fraction ratios of 44 elements relative to the mass fraction of calcium for the diagnosis of PCa.

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 Seminal Ratios of

fraction fluid means

BPH to PCa to PCa to ratio Normal BPH PCa

Normal Norma! BPH

Ca/Li 82584 48711 109809 0.6 1,3 2.3

Ca/B 3801 1967 300 0.5 0.3 0.7

Ca/Na 0.1 0.2 0.04 1.4 0.3 0.2

Ca/ g 2.8 2.2 9.3 0.8 3.4 4.1

Ca/AI 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 30000 3.0 2.0 0.7

Ca Cr 7331 15114 079 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 1 9.8 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 22 025 823700 30000 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 131 31 95502 0.6 0.4 0.7

Ca/Ba 22721 32499 9663 1.4 0.4 0.8

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/T 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 34556 116232 130000 0.9 1.0 1. Ca/Cd 252833 324398 68768 1.3 0.3 0.2

Ca/Hg 84303 164740 130000 2.0 1.5 0.8

Ca/T! 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 el tiv to the mass raction of zinc ^" m seminal fluid derived from normal, cancerous and BPH subjects.

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 norma! seminai fluid, BPH and cancerous seminai 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 10. Means of ratios and their ratios between mean values of mass fraction ratios of Zn to mass fractions of other chemical elements in seminai fluid derived from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects.

Seminal Ratios of

ass fraction

fluid means

BPH to PCa to PCa to ratio Norma! BPH PCa

Normal Norma! BPH

ZniU ^" Ϊ989& 23655 11826 1.19 0,59 0.50

Zn/8 916 955 140 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/A! 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 Ζπ/Ou 338 316 142 0.93 0.42 0.45

Ζπ/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 66.2 28.0 1.60 0.27 0.17

Zn/Sr 1612 1755 283 1.09 0.18 0.16

2n Y 53253 400000 14000 7.51 0.26 0.04

Zn/Zr 9490 53209 3072 5.81 0.32 0.06

Zn/Ag 73100 400000 4680 5;47 0.06 0.01

Zn/S 7310 40000 14000 5.47 1.92 0.35

Zn Cs 52970 83970 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 6091 157532 7406 2.59 0.12 0.05

Zn/Hg 20312 80000 14000 3.94 0.69 0.18

Zn/T! 73100 400000 14000 5.47 0.19 0.04

Zn/Pb 7392 28456 1857 3.85 0.22 0.06

Zn/Bi 58072 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

E ample 29, Using the Ca/Mn. mass fraction ratio In EPS to establish prostate condition.

The Ca/Mn mass fraction ratio in EPS was found to be signifieantiy different in most cancerou EPS as compared to normai 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). if PCa needs to be discriminated from normai and BPH and if the Ca/Mn ratio in the EPS sample prepared and analysed as described in Example 17 does not exceed 900, prostate carcinoma with an accuracy exceeding 96% can be diagnosed.

Example 30. Using the Ca/A! mass fraction ratio in seminal fluid to estabiish prostate The Ca/AI mass traction ratio in seminal fluid was found to be significantly different in most cancerous seminal fiuid as compared to normal and benign hyperplastic seminal fluid. The upper limit for Ca/AI mass fraction ratio on dry mass basis in cancerous seminal fluid was determined to be 290 (Table 9). if PCa seminai fluid needs to be discriminated from normal and BPH one and if the Ca/AI 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.

Exam e 31. Using the Zn/Cu mass fraction ratio in EPS to establish prostate condition.

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).

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. Usin§ the Zn/Cu mass fraction ratio in seminai fluid to establish prostate condition.

The Zn/Cu mass fraction ratio in seminai fiuid 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 fractio ratio on dry mass basis in cancerous seminal fluid was determined to be 155 (Tabie 10). 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*¾fg*Zn/IVln*Bi*Se mass fraction ratio combination in EPS to establish prostate condition.

The Ca* g*Zn/Mn*Bt*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/IVin ^'A' Bi*Se mass fraction ratio on dry mass basis in healthy EPS was determined to be 2E8. If PCa EPS needs to be discriminated from norma! and BPH EPS and if the Ca* g*Zn/Mn*Bi*Se ta: o in the EPS sample prepared and anaiysed as described in Example 18 is below 2E8, prostate carcinoma with an accuracy better than 95% can be diagnosed,

Example 34. Using the C.a*Mg*Zn/$In*Bi*Se mass fraction rati© combination in seminal fluid to establish prostate condition.

The Ca* g*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* g*2n/ n ^'A Bi*Se mass fraction ratio on dry mass basis in healthy seminal fluid was determined to be 2E6, If PCa seminal fluid needs to be discriminated from normal and BPH seminai fluid and if the Ca*iVSg*Zn/ n*Bi*Se ratio in the seminai fluid sample prepared and anaiysed as described in Example 19 is below 2E6, prostate carcinoma with an accuracy better than 95% can be diagnosed.

Exampie 35. Using the Ca Ba mass fraction ratio to establish prostate condition,

The Ca/8a mass fractio ratio was found to be significantly different in most cancerous prostate tissues as compared to normal and benign hyperpiastic tissues (Exampie 8, Table 2}, The uppe limit for Ca/Ba mass fraction ratio on dry mass basis in cancerous prostate tissue was determined to be +3SD (M - arithmetic mean, SD - standard deviation) or 400 (Figure 13). if PCa needs to be discriminated from norma! and BPH tissue and if the Ca/Ba ratio in a prostate biopsy sample prepared and anaiysed as described in Exampie 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 hased test is 100-9% and 100-2%, respectively. Exampie 38, Using the Ca/P mass fraction ratio to establish prostate condition.

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 (Exampie 8, Table 2). The upper limit for Ca/P mass fraction ratio on dry mass basis i cancerous prostate tissue was determined to be M+3SD (M - arithmetic mean, SD - standard deviation) or 0. 5 (Figure 4). if PCa needs to be discriminated from normal and 8PH 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 _f prostate carcinoma with an accuracy of 98±2% can be diagnosed. The sensitivity and specificity of the Ca/P ratio based test is 91 ±9% and 100-3%, respectively.

Example 37. Using the Ca/Si mass f raction ratio to establish prostate condition. The Ca/Si mass fraction ratio was found to be significantly different in most cancerous prostate tissues as compared to norma! 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+3SO ^' .{M - arithmetic mean, SD - standard deviation) or 5 (Figure 16). if PGa 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 98±2% can be diagnosed. The sensitivity and specificity of the Ca Si ratio based test is 91 ±9% and 100-3%, respectiveiy.

Example 38. Using the Ca/Sr mass fraction ratio to establish prostate condition.

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 +3SD ( - arithmetic mean, SD - standard deviation) or 250 (Figure 16), if PCa needs to be discriminated from norma! 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 98±2% can be diagnosed. The sensitivity and specificity of the Ca/Sr ratio based test is 91 ±9% and 100-3%, respectively.

Example 39. Using the Zn/Mn mass fraction ratio to establish prostate condition.

The Zn/Mn mass fraction ratio was found to be significantly different In most cancerous prostate tissues as compared to nomnal and benign hyperplastic tissues (Example 8, Tafoie 2}. The upper limit for Zn/Mn mass fraction ratio on dry mass basis in cancerous prostate tissue wa determined to be M+3SD (M - arithmetic mean, SD - standard deviation) or 170 (Figure 17). if PCa needs to be discriminated from norma! 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 98±2% can be diagnosed. The sensitivity and specificity of the Zn/Mn ratio based test is 91 ±9% and 100-3%, respectively.

Example 40. Using the [CZn*Ca*Mg*Cd)/{S Br*Af*Ba}]*10O0 mass fraction ratio combin ion to establish prostate condition. The t(Zn a*Mg*Cd}/(Si*Br*A!*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 ^* i./lg ^* Cd)/{Si ^i: Br*A Ba}]*1000 mass fraction ratio combination on dry mass basis in cancerous prostate tissue was determined to be +60SD (M ~ arithmetic mean, SD - standard deviation) or 100 000 (Figure 8}. If PCa needs to be discriminated from norma! and BPH tissue and if the [{Zn ^* Ca*IV!g*Cd)/(Si ^* Br ^* Ai ^* Ba)]* 000 mass fraction ratio combination in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 100 000, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the [{Zn ^* Ca* g*Cd) (Si*a-*A[ ^* 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, Ai _s Ba,8l, Br,Ca, C<£, Ce, Co, Cr, Cs, Cu, Hg, K, Π, Mg, Mn, Ma, Hi, P, Pb _s fa, S, Sb, Se, SI, St nd Zn to establish prostate condition from th® prostate tissue samples .

Mass fractio 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.

The following combination of normalised mass fraction ratios [{C¾ ^* C¾ _n cto*Hg _n *Mg _n *Na _n ^

i _n *Mnn*Nin*Pbn*Sbn*S ^' in*Sr _n )]*10 ^1i> was found to be significantly different in most cancerou prostate tissues as compared to normal and benign hyperplastic tissues. The upper limit for in ^* n _n *Nin*Pbr _l *Sbn*Sin*Srn)3*10 ¹⁸ combination of normalised mass fraction ratios on dry mass basis in cancerous prostate tissue was determined to be 00 000 000 000 (Figure 19). if PCa needs to be discriminated from normal and BPH tissue and if the

in*Mnn* in ^:* Pb _t i ^* Sbn ^'A Sin ^: *Srn)]*10 ^1s ratio in a prostate biopsy sample prepared and analysed as described in Example 1 does not exceed 100 000 000 000, prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the

i _B ^* Mn _n *Nia*Pb„*Sbn*Sin*Sr _n )]*10 ¹⁸ ratio based test is 100-10% and 100-3%, respectively.

Example 42. Using the norm lised mass fraction ratio combinations of Ag, Al _» A , B, 8a, Bl, 8r _s Ca, Cd, Ce, Co, Cr, Cs,€u _s Oy _? Er, Fe _s Gel, Hg, Ho, K, La, Li, M , n, Na, N _s fcji, P, Pb, Pr, Rb, S, Sfa, Sc, Se, St, Sm, Sr _y T , T!, U, Y, Zsi arsd Zr to establish prostate condition from the prostate tissue samples.

Mass fraction levels of the elements can be normaiized to the reference levels of same elements. Further, combination of normalized mass fraction ratios can be used to diagnose prostate condition. To improv 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, Ai, 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, i, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, St., 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. The

[(Ca„ ^* Cd _n *Co ₀ *Hg _n ^* K^

^* Zr _n }]*10 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 sampies. 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 (Figure 20). The upper limit for

*Zr. )] ^'v 1 Q ^M normalized mass fractio ratio combination on dry mass basis in cancerous prostate tissue was determined to be 10 ²⁴ (Figure 20). If PCa needs to be discriminated from normal and BPH tissue and if the

^* Zr _n }] ^* 10 ³⁴ normalised mass fraction ratio combination in a prostate biopsy sample prepared and analyzed as described in Example 1 does not exceed 10 ^a _f prostate carcinoma with an accuracy of 100-2% can be diagnosed. The sensitivity and specificity of the n*CSn*Cu _n ^* Dy Er _n e _r *G n*Hon*Lan ^

^* Zr _n )] ^* 10 ³ normalized mass fraction ratio combination based test is 100- 0% and 100-3%, respectively.

Example 43. Identification of cancer foiomarkes in expressed prostatic secretion using Inductively Coupled Plasma ifess Spectrometry {lCP-iVSS} and Inductively Coupled Atomic Emission; Spectrometry {IGP-AES}.

Equipment; Inductively coupled plasma mass spectrometry instrument Agilent 7500c, Specimen:

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 (TRUSS) and microscopic analysis of tissue morphology in biopsies obtained from the sam patients, where prescribed by the referring physician.

Reagents:

HN03 (nitric acid 65 % for analysis, max. 0.005 ppm Hg, GR, ISO, Merck), H2O2 (hydrogen peroxide pure for analysis, Merck), !CP~MS standards NCSZC73013 (NCS Certified

Reference Material), BCR063R (Community Bureau of Reference of the European

Comission) and IR BD151 (LGC Standards, Weisel, Germany). ProtocGl:

0-5 mL of HNO3 was added to freeze-dried EPS samples and the samples were left over night at room temperature. After that 0.25 mL of HNO and 0,15 mL of H2O2 were added to the samples and piaced 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 bottie. Simultaneously, the same procedure was performed on a sample containing no EPS fluid, and the resultant solution was used as a blank sample. Ail samples were analyzed by Inductively Coupled Plasma Mass

Spectrometry and inductively Coupled Plasma Atomic Emission Spectrometry'. The spectrometer parameters and the main parameters of !CP-MS measurements: auxiliary air fiow rate - 0.9 L/min, nebulizer flow rate - 0.9 L/min, sample update - 0.8 mL/min. The spectrometer parameters for iCP-AES measurements; generator output power 1 ,500 VV.

Results:

The content of Al, Cd, Cs, Mn, Ni, Rb, S, Se and Si in EPS was analyzed by SCP-MS, The content of Na, Mg, P, S, K, Ca, Fe, Cu, 2n and Ba in EPS was analyzed by ICP-AES,

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

Differences in mass fraction leveis 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 11. Comparison of median values of chemical element mass fractions (mg kg , dry mass basis) in normal, benign hyperplastic (BPH) and iow grade cancerous (PCa) EPS.

Normal BPH PCa BPH/Normai PCa/Norma! PCa/BPH

Ai 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 5.2? 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 βδΟΟ 1 .0 1 .4 1 .4

Mn 0.50 0.93 1.58 1 .8 3.1 1 .7

Ha 41286 51010 47000 1 .2 1 .1 0.9

Ni 0.43 0.83 0.73 1.9 17 0.9

P 3350 3800 4400 1.1 1.3 12

R 40.75 30.93 47.66 0.8 12 1.5

S 5809 8400 10521 1.1 1.8 1 .6

S 1.24 1.26 1 .97 1 .0 1 .6 16

Si 84.32 105 4 10.0 13 13 1.0

Zn 5135 4100 8000 0.8 1.6 2.0

Exampl© 44. Peterrnmaitors of normalised mass fraction levels of elements In n rmal, BPH and low grade adenocarcinom EPS.

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 ievels of the same elements are presented. Reference levels in this example represent mean ievel values derived from the group of 10 EPS samples from verified healthy volunteers. Anybody skilled in the field can appreciate that corresponding reference Ievels must be determined for different patient populations. Table 12. Mean mass fraction levels of elements normalized to the reference ievels of th same elements in normal, BPH and iow grade adenocarcinomatous EPS.

Example

Reference

levels,

mg/kg dr

mass

basis Normal BPH Pea BPH/Nor ai PCa/Norrnal PCa/BPH

Al 33.89 0.9 1 .1 2.6 1.1 2,6 2.4

Ba 4.27 10 11 3.3 1.1 3.3 3.1

Ca 9528 1 .1 1 .0 15 1.0 15 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 11 16

Cu 7.3 10 12 1 .3 1 .2 1.3 1 .1

Fe 44.66 0.9 1.4 1 .0 14 10 0.8

32402 1.1 1 .0 1.6 10 1 .6 17

Mg 4225 1.1 10 1.6 1.0 1 .6 15

Mn 0.94 1 .0 1.8 15 1.8 15 0.8 Ha 39362 1.0 1.5 1.5 1.5 1.5 1.0 \ 0.74 0,9 2.6 1.5 2.6 1.5 0,6

P 3901 0.9 1.1 1.0 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 1.8 1 ,5

Se 1.52 1.0 0.9 1.4 0,9 1 ,4 1.6

Si 75.41 0.9 1.6 2,8 1.6 2.6 1.6

Z 4870 1 ,0 0.8 1 ,7 0,8 1.7 2.1

The data in the Table 2 allow evaluating the importance of normalize mass fraction levels for the diagnosis of PCa. To illustrate this further examples are given. mple 4§„ Establishing the prostat® condition using the additive inde based on the normad^ecl mass fractions of Ca, K, Mg and Zn in EPS.

Further, based on the normalized mass fractions of the elements determined as described in Example 44 the Additive index (A!) of prostate condition can be calculated, as exemplified here: where Ca,¾, K _n , Mg _n , 2n„ represent normalised vaiues, i.e. mass fractions of Ca, K, Mg and Zn in EPS samples of the subject, divided by the reference ieveis of the same elements. Additive index was found to be significantly different i most cancerous EPS as compared to normal and benign hyperplastic EPS (Table 13).

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

TabS© 13 Comparison of the Additive Indices between the diagnostic groups.

N iwor» m tuarnl BPH PCa

-3e-0Q7 -0.21 2.0

Std. Deviation 12 1 .0 1.1

Std. Error of Mean 0.39 0.28 0.49

Lower 95% Ci of mean -0.9 -0.8 0.6

Upper 95% Ci of mean 0.9 0.4 3.3 Exarapie 48. Establishing the prostate ^. condition using the multiplicative Index based on the normalized mass fractions of Ca, K, Mg and Zn In EPS.

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

Ml ~ (C¾,*Kn*Mg _n *Zn„)/4 where C¾ 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 (Tabl 14).

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

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% Ci of mean 0.2 0, 1 0.1

U per 95% CI .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, b, S and Zn in EPS.

Further, based on the normalized mass fractions of the elements determined as described in Example 44 the 6-e!ement Multiplicative index ( !/δ) of prostate condition can be calculated, as exemplified here: Mi/6 ⁱ = (Can*KB*Mg _n *Rbn*S *Znn) 6 where Ca _n ,Kn, gn, Rb _n ,S« and Zn _n represent mass fractions of Ca _r 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). if PCa needs to be discriminated from normal and BPH and if the Multiplicative Inde 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 (Figure 23). Table 15. Comparison of the Multiplicative indices between the diagnostic groups.

Normal BPH PCa

Mean 0.3 0.2 4.1

Sid. Deviation 0.3 0.13 6.4

Std. Error of Mean 0.1 0,04 2.9

Lower 95% CI of mean 0.13 0.07 -3.9

Upper 95% CI of mean 0.6 0.2 12

Exampi© 48. Identification of cancer blomarkes In expressed prostatic secretion using Energy Dispersive X-Ray Fluorescence (EOXRF),

Equipment and method: EDXRF spectrometer consisted of an annular ¹⁰⁹ Cd source with an activity of 2,56 GBq, a 25 mm ² Si(Li) detector and portable multichannel ana!yzer combined with a PC, its resolution was 270 eV at the 5.9 keV line of 55Fe~souree. The duration of the Zn measurements together with Br, Fe, R _s and Sr was 80 min. The intensity of Ka-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 Ka-lines for samples and standards.

Specimen:

Expressed Prostatic Secretion samples (EPS) from patients with Benign Prostate Hyperplasia (BPH} and adenocarcinoma (PCa) and EPS samples from healthy volunteers were obtained by trarisrectai 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. Sample preparation:

20 μΙ of the EPS sample were placed on a backing com prised of a thin film of transparent polymeric materia! (Dacron, Mylar, polyethylene or similar, thickness < 10,um). The drop of a sampl was freeze-dried on a backing until the constant mass. Results:

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.

Differences in mass fraction levels of Zn and Rb were found to be statistically significant in samples derived from cancerous, benign hyperplastic and normai EPS samples. 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}/1G was found to be the most informative marker of prostate cancer. The data in Tabie 16 allow evaluating the importance of the combination of mass fraction levels of Rb and Zn for the diagnosis of clinical prostate cancer (PCa). 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 (Figure 24).

Table 18, Parameiers of the importance (sensitivity, specificity and accuracy) of the Product index (Rb*Zn)/ 0 in the sam ies 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