Research Article

Distinguishing Malignant from Benign Prostate Tumors using Br, Fe, Rb, Sr, and Zn Content in Prostatic Tissue

Rossmann M¹, Zaichick S² and Zaichick V^3*
1Department of Biochemistry, University of Cambridge, UK
²Department of Medicine, University of Illinois College of Medicine, USA
³Department of Radionuclide Diagnostics Medical Radiological Research Centre, Russia

^*Corresponding author: Zaichick V, Department of Radionuclide Diagnostics Medical Radiological Research Centre, Russia

Published: 13 Jul 2016
Cite this article as: Rossmann M, Zaichick S, Zaichick V. Distinguishing Malignant from Benign Prostate Tumors using Br, Fe, Rb, Sr, and Zn Content in Prostatic Tissue. Clin Oncol. 2016; 1: 1054.

Abstract

Contents of Br, Fe, Rb, Sr, and Zn in normal (n=37), benign hypertrophic (n=43) and cancerous tissues (n=60) of the human prostate gland were investigated by radionuclide-induced (109Cd) energy dispersive X-ray fluorescent (EDXRF). Mean values (M±SΕΜ) for mass fraction (mg/kg on dry mass basis) of trace element in the normal tissue were as follows: Br 40.6±5.6, Fe 118±8, Rb 16.3±1.1, Sr 2.5±0.4, and Zn 1154±119, respectively. Mean values (M±SΕΜ) for ratio of mass fractions were: Zn/Br 39.1±6.2, Zn/Fe 11.2±1.3, Zn/Rb 71.7±9.0, and Zn/Sr 534±83, respectively. It was observed that in benign hypertrophic tissues the levels of Br, Fe, Rb, Sr, and Zn were equal to those in normal prostate tissues. By contrast, the levels of Rb and Zn were significantly lower and those of Br and Sr were significantly higher in cancerous tissues than in normal tissues. The Zn mass fraction, as well as Zn/Br and Zn/Sr ratios, were the most informative indicators for distinguishing malignant from benign prostate with sensitivity, specificity, and accuracy in the ranges 95-100%, 92-98%, and 95-98%, respectively. Obtained data allowed us to adequately evaluate the importance of trace element content and their ratios for the diagnosis of prostate cancer.
Keywords: Zinc; Trace elements; Intact prostate; Benign prostatic hyperplasia; Prostate cancer; Energy-dispersive X-ray fluorescent analysis

Introduction

The prostate gland may be a source of many health problems in past middle age men, the most common being benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCa). BPH is a noncancerous enlargement of the prostate gland leading to obstruction of the urethra and can significantly impair quality of life [1]. The prevalence of histological BPH is found in approximately 50-60% of males age 40-50, in over 70% at 60 years old and in greater than 90% of men over 70 [2,3]. In many Western industrialized countries, including North America, PCa is the most frequently diagnosed form of noncutaneous malignancy in males and, except for lung cancer, is the leading cause of death from cancer [4-9]. Approximately one in eight men will be diagnosed with prostate cancer at some point of their lifetime. Although the etiology of BPH and PCa is unknown, many trace elements have been highlighted in the literature in relation to the development of these prostate diseases [10-29]. Trace elements have essential physiological functions such as maintenance and regulation of cell function and signalling, gene regulation, activation or inhibition of enzymatic reactions, neurotransmission, and regulation of membrane function. Essential or toxic (mutagenic, carcinogenic) properties of trace elements depend on tissue-specific need or tolerance, respectively [30]. Excessive accumulation, deficiency or an imbalance of the trace elements may disturb the cell functions and may result in cellular degeneration, death and malignant transformation [31]. In previous study significant changes of trace element contents in hyperplastic and cancerous prostate in comparison with those in the normal prostatic tissue were observed [32-53]. Moreover, a significant informative value of Zn content as a tumor marker for PCa diagnostics was shown [54]. Hence it is possible that besides Zn, some other trace elements also can be used as tumor markers for distinguish between benign and malignant prostate. Current methods applied for measurement of trace element contents in human tissue samples include a number of techniques. Among these methods the energy dispersive X-ray fluorescence technique (EDXRF) is one of the most simple, fast, reliable, efficient, and available techniques. There are many different kinds of EDXRF devises on the market and technical possibilities of this method improve rapidly. Therefore, the present study had had three aims. The main objective was to assess the Br, Fe, Rb, Sr, and Zn contents as well as Zn/Br, Zn/Fe, Zn/Rb, and Sr/Zn ratios in intact prostate of healthy men aged over 40 years and in the prostate gland of age-matched patients, who had either BPH or PCa using ¹⁰⁹Cd-radionuclide induced EDXRF analysis. The second aim was to compare the levels of trace elements in normal, hyperplastic, and cancerous prostate, and the third aim was to evaluate the trace element content for diagnosis of prostate cancer.

Table 1

Table 2

Material and Methods

All patients studied (n=103) were hospitalized in the Urological Department of the Medical Radiological Research Centre. Transrectal puncture biopsy of suspicious indurated regions of the prostate was performed for every patient, to permit morphological study of prostatic tissue at these sites and to estimate their chemical element contents. In all cases the diagnosis has been confirmed by clinical and histopathological assessment of biopsy and surgically resected materials. A histological examination in the control group was used to establish the age norm, as well as to confirm the absence of microadenomatosis and latent cancer.
The age of 43 patients with BPH ranged from 38 to 83 years, the mean being 66±8 years (M±SD). The 60 patients aged 40-79 suffered from PCa. Their mean age was 65±10 years. Intact prostates were removed at necropsy from 37 men (mean age 55±11 years, range 41-79) who had died suddenly. The majority of deaths were due to trauma.
Tissue samples were divided into two parts. One part of samples was used for histological and the other for trace element analysis. For the purpose of the latter samples were weighed, freeze-dried and then homogenized. The pounded sample weighing about 8 mg was applied to the piece of Scotch tape that served as an adhesive fixing backing. To determine the contents of the elements by comparison with a known standard, aliquots of commercial, chemically pure compounds were used. The microliter standards were placed on disks made of thin, ash-free filter papers fixed on the Scotch tape pieces and dried in a vacuum. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) weighing about 8 mg were analyzed to estimate the precision and accuracy of results. The CRM IAEA H-4 subsamples were prepared in the same way as the samples of dry homogenized prostate. Details of the relevant facility for EDXRF, source with ¹⁰⁹Cd radionuclide, methods of analysis and the results of quality control were presented in our earlier publications concerning the EDXRF analysis of human prostate tissue [17,55]. All prostate samples were prepared in duplicate and mean values of trace element contents were used in final calculation. Using the Microsoft Office Excel software, the summary of statistics, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for trace element contents in normal, benign hyperplastic and cancerous prostate. The same programs were used to estimate the inter-correlations of trace element contents. The reliability of difference in the results between the three groups of prostate samples was evaluated by Student’s t-test.
The study was approved by the Ethical Committees of the Medical Radiological Research Centre, Obninsk.

Table 3

Table 4

Results

Table 1 depicts our data for 5 trace elements in ten sub- samples of CRM IAEA H-4 (animal muscle) and the certified values of this material.
Table 2 presents certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the Br, Fe, Rb, Sr, Zn mass fraction and the Zn/Br, Zn/Fe, Zn/Rb, Zn/Sr mass fraction ratios in normal, benign hypertrophic and cancerous prostate. The ratios of means and the reliability of difference between mean values of Br, Fe, Rb, Sr, Zn mass fractions and the Zn/Br, Zn/ Fe, Zn/Rb, Zn/Sr mass fraction ratios in normal, benign hypertrophic and cancerous prostate are presented in Table 3.
The comparison of our results with published data for Br, Fe, Rb, Sr, and Zn mass fraction in normal, benign hypertrophic and cancerous prostate is shown in Table 4.
Figure 1 and 2 depict individual data sets for Br, Fe, Rb, Sr, Zn mass fraction and for the Zn/Br, Zn/Fe, Zn/Rb, Zn/Sr mass fraction ratios, respectively, in all samples of normal, benign hypertrophic and cancerous prostate.

Discussion

As was shown by us [17,55] the use of CRM IAEA H-4 as a CRM for the analysis of samples of prostate tissue can be seen as quite acceptable. Good agreement of the Br, Fe, Rb, Sr, and Zn contents analyzed by EDXRF with the certified data of CRM IAEA H-4. Table 1 indicates an acceptable accuracy of the results obtained in the study of trace elements of the prostate presented in Tables 2-4. The mean values and all selected statistical parameters were calculated for 5 trace elements (Br, Fe, Rb, Sr, and Zn) and for 4 ratios (Zn/Br, Zn/ Fe, Zn/ Rb, and Zn/Sr) of chemical element mass fractions (Table 2). The mass fraction of Br, Fe, Rb, Sr, and Zn were measured in all, or a major portion of normal, BPH and PCa samples. From Table 3, it is observed that in benign hypertrophic tissues the mass fractions of Br, Fe, Rb, Sr, and Zn not differ from normal levels. In cancerous tissue the mass fractions of Rb (p<0.01) and Zn (p<0.001) are lower, and mass fractions of Br (p<0.01) and Sr (p<0.01) are significantly higher than in normal tissues of the prostate. Except for Fe and Sr, the mass fractions of all the other elements show significant variations in cancerous tissues when compared with benign hypertrophic tissues of the prostate. The mass fractions of Rb (p<0.01) and Zn (p<0.001) are lower and mass fraction of Br (p<0.01) is higher than in benign hypertrophic tissues. The mean level of Zn/Br mass fraction ratio in BPH is significantly (p<0.05) higher than in normal prostate. In cancerous tissue the Zn/Br, Zn/Fe, Zn/ Rb, and Zn/Sr mass fraction ratios are significantly (p<0.001) lower than in normal and benign hypertrophic tissues. The results for all trace element contents in the prostates of the control group (mean age 55±11 years, range 41-79) are in accordance with our earlier findings in prostates of apparently healthy men aged 41-60 [17]. Values obtained for Br, Fe, Rb, Sr, and Zn contents (Table 4) agree well with median of mean values cited by other researches for the human prostate [32-44]. Data of the literature also includes samples obtained from patients who died from different diseases. A number of values for chemical element mass fractions were not expressed on a dry mass basis in the cited literature. Therefore, we calculated these values using published data for water - 80% [43] and ash - 1% on wet mass basis [56] contents in the prostate of adult men. Our results for Br, Fe, and Zn are in accordance with the medians of earlier findings in benign hypertrophic tissues of prostate (Table 4). No published data referring Rb and Sr contents in benign hypertrophic tissues of the prostate were found. In cancerous prostate tissues our results were comparable with published data for Fe and Zn contents and two orders of magnitude higher for Br (Table 4). No published data referring Rb and Sr contents of cancerous prostate tissue were found. In BPH the mean Br, Fe, Rb, Sr, and Zn mass fractions were equal to normal prostate (Table 3). This result is in accordance with earlier findings for Fe [34,57,58] and for Zn [34,35,39,43,57-69]. In cancerous tissues mean values for Br and Sr mass fractions were significantly higher whereas mean values for Rb and Zn mass fractions were significantly lower than in normal prostate (Table 3). Data obtained for Zn are in agreement with previously reported findings for this element [35,39,40,57-61,63-65,67-71]. In cancerous tissues mean values for Br, Fe, and Sr mass fractions were equal to or greater than those for benign hypertrophic prostate tissues and Rb and Zn mass fractions were significantly lower (Table 3). This result is in accordance with earlier findings for Fe [35,41,58,72] and for Zn [35,39,41,43,44,57,60,61,63-69,71,73-78]. No published data referring to mass fraction ratios of Br, Fe, Rb, Sr, and Zn in normal, benign hypertrophic and cancerous prostate could be found.
Characteristically, elevated or deficient levels of trace elements observed in cancerous tissues are discussed in terms of their potential role in the initiation, promotion, or inhibition of prostate cancer. In our opinion, abnormal levels of many trace elements in cancerous tissue could be the consequence of malignant transformation. Compared to other soft tissues, the human prostate has higher levels of Zn, Ca, Mg and some other trace elements [16,17,79-83]. These data suggests that these elements could be involved in functional features of prostate tissue [84-88]. Malignant transformation is accompanied by a loss of tissue-specific functional features, which leads to a significant reduction in the contents of elements associated with functional characteristics of the human prostate tissue (Zn and, probably, Rb – (Table 3). Therefore, it is plausible that the reason for the emergence and development of cancer is associated with abnormally high concentration of some metals in the prostate tissue of older men [16,17,20-24]. The same reasoning could be applied to benign hypertrophic tissue (it retains functional features of normal prostate tissue) and its increased levels Sr and Zn (Table 3). Trace elemental analysis of prostate tissue could become a powerful diagnostic tool. To a large extent, the resumption of the search for new methods for early diagnosis of PCa was due to experience gained in a critical assessment of the limited capacity of the serum prostate specific antigen (PSA) test [89]. In addition to the PSA serum test and morphological study of needle-biopsy cores of the prostate, the development of other highly precise testing methods seems to be very useful. Experimental conditions of the present study were approximated to the hospital conditions as closely as possible. In all cases we analyzed a part of the material obtained from a transrectal puncture biopsy of the indurated site in the prostate. Therefore, our data allow us to evaluate adequately the importance of trace element content information for the diagnosis of PCa.
Tissue content of Br, Rb, and Zn are significantly different in most cancerous tissues as compared to normal and benign hypertrophic tissues (Table 3). As is evident from individual data sets (Figure 1), the Zn mass fraction is one of the most informative for a differential diagnosis. If 350 mg/kg dry tissue (M±2.5SD) is the value of Zn mass fraction assumed to be the upper limit for PCa (Figure 1) and an estimation is made for “PCa or intact and BPH tissue”, the following values are obtained:
Sensitivity = {True Positives (TP) / [TP + False Negatives (FN)]} ·100% = 98±2%;
Specificity = {True Negatives (TN) / [TN + False Positives (FP)]} ·100% = 92±3%;
Accuracy = [(TP+TN) / (TP+FP+TN+FN)] ·100% = 95±2%.
The number of people (samples) examined was taken into account for calculation of confidence intervals [90]. In other words, if Zn contents in a prostate biopsy sample do not exceed 350 mg/kg dry tissue, one could diagnose a malignant tumor with an accuracy of 95±2%. Thus, using the Zn-test makes it possible to diagnose cancer in 98±2% cases (sensitivity). We have previously shown that not only the absolute values of the chemical elements can be used successfully for diagnostic purposes but also their ratios and other mathematic combinations [91]. The use of the relations between mass fractions of chemical elements is particularly promising for the development of in vivo diagnostic methods, including the diagnosis of PCa. As is evident from individual data sets (Figure 2), the Zn/Br and Zn/Sr mass fraction ratios are the most informative indicators for a differential diagnosis. If 2.9 (M±2.5SD) and 40 (M±2.5SD) are the values of Zn/Br and Zn/ Sr mass fraction ratios assumed to be the upper limit for PCa (Figure 2) and an estimation is made for “PCa or intact and BPH tissue”, the following values are obtained: using Zn/Br ratio - sensitivity 95±5%, specificity 98±2%, and accuracy 97±2%; using Zn/Sr ratio - sensitivity 100-10%, specificity 982%, and accuracy 98±2%. It should be noted, however, that Br is a component of many tranquilizers. It is possible that the increase in Br content could be explained by uncontrolled use of tranquilizers in the group of PCa patients. Therefore, for diagnostic purposes, data for Br content should be used with caution. Possibility of using the Zn/Sr ratio is limited by difficulties in determining the Sr content by EDXRF because the content of this element in the prostate tissue is close to the limit of detection.

Figure 1

Conclusion

In this work, trace elemental analysis was carried out in the tissue samples of normal, benign hypertrophic, and carcinomatous prostates using EDXRF. It was shown that EDXRF is an adequate analytical tool for the non-destructive determination of Br, Fe, Rb, Sr, and Zn content in the tissue samples of human prostate, including needle-biopsy cores. It was observed that in benign hypertrophic tissues the contents of Br, Fe, Rb, Sr, and Zn were equal to those in normal prostate tissues. It is possible that elevated levels of Sr and Zn initiate and promote prostate cancer by oxidative DNA damage, which is caused by an increase in generation of free radicals and a decrease in the antioxidant defense capacity of cells. The contents of Rb and Zn were significantly lower and those of Br and Sr were significantly higher in cancerous tissues than in normal tissues. In our opinion, the abnormal decrease in levels Rb and Zn in cancerous tissue could be a consequence of malignant transformation. Finally, we propose to use the Zn mass fraction in a needle-biopsy core as an accurate tool to diagnose prostate cancer. Further studies on larger number of samples are required to confirm our findings, to study the impact of the trace elements on prostate cancer etiology and to examine the long-term pathological outcome.

Figure 2

Acknowledgements

We are grateful to Dr. Tatyana Sviridova, Medical Radiological Research Center, Obninsk, and to the late Prof. A.A. Zhavoronkov, Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow, for supplying prostate samples.

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