DIAGNOSIS OF THYROID MALIGNANCY USING TRACE ELEMENTS OF NODULAR TISSUE DETERMINED BY X-RAY FLUORESCENCE ANALYSIS 

Vladimir Zaichickimage 

Radionuclide Diagnostics Department, Medical Radiological Research Centre, Obninsk, 249036, Russia.

ABSTRACT 

Background: Benign (TBN) and malignant (TMN) thyroid nodules are a common thyroid lesion. The differentiation of TMN often remains a clinical challenge and further improvements of TMN diagnostic accuracy are warranted. The aim of this study is to evaluate the possibilities of using differences in trace element contents (TEs) in nodular tissue to diagnose thyroid malignancies and to determine the sensitivity, specificity, and accuracy for most informative TEs in the diagnosis of TMN.

Methods: Contents of TEs such as bromine (Br), copper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn) were prospectively evaluated in “normal” thyroid (NT) of 105 individuals as well as in nodular tissue of thyroids with TBN (79 patients) and to TMN (41 patients). Measurements were performed using energy-dispersive X-ray fluorescent analysis.

Results: It was observed that in TMN tissue the mean mass fractions of I and Zn were lower while the mean mass fraction of Rb was higher than in NT and TBN tissue. It was demonstrated that the I contents is nodular tissue is the most informative parameter for the diagnosis of thyroid malignancy. It was found that “Sensitivity”, “Specificity” and “Accuracy” of TMN identification using the I level in the needle biopsy of affected thyroid tissue (87±5%, 96±2% and 94±2% respectively) were significantly higher than that made using ultrasound screening and cytological test of fine needle aspiration biopsy. 

Conclusions: It was concluded that determination the I level in a needle biopsy of TNs using energy-dispersive X-ray fluorescent analysis, is a fast, reliable, and informative diagnostic tool that can be successfully used as an additional test of thyroid malignancy identification.

Keywords: Diagnosis of thyroid malignancy, energy-dispersive X-ray fluorescent analysis; normal thyroid; thyroid nodules; trace elements.

 

INTRODUCTION

 

Nodules are a common thyroid lesion, particularly in women. Depending on the method of examination and general population, thyroid nodules (TNs) have an incidence of 19–68%1. In clinical practice, TNs are classified into benign (TBN) and malignant (TMN), and among all TNs approximately 10% are TMN2. It is appropriate mention here that the incidence of TMN is increasing rapidly (about 5% each year) worldwide2. Surgical treatment is not always necessary for TBN whereas surgical treatment is required in TMN. Thus, differentiating TBN and TMN will have a great influence on thyroid therapy.

Ultrasound screening (USS) is widely used as the primary method for early detection and diagnosis of the TNs. However, there are many similarities in the USS characteristics of both TBN and TMN. For misdiagnosis prevention some computer-diagnosis systems based on the analysis of USS images were developed, however as usual these systems for the diagnosis of TMN showed accuracy, sensitivity, and specificity nearly 80%2,3. Therefore, when USS examination shows suspicious signs, an US-guided fine-needle aspiration biopsy is advised. Despite the fact thatfine needle aspiration biopsy has remained the diagnostic tool of choice for evaluation of USSsuspicious thyroid nodules, the differentiation of TMN often remains a diagnostic and clinical challenge since up to 30% of nodules are categorized as cytologically “indeterminate”4. Thus, to improve diagnostic accuracy of TMN, new technologies have to be developed for clinical applications. However, a recent systematic review and meta-analysis of molecular tests in the preoperative diagnosis of indeterminate TNs has shown that presently there is no perfect biochemical, immunological, and genetic biomarkers to discriminate malignancy5. Therefore, further improvements of TMN diagnostic accuracy are warranted. During the last decades it was demonstrated that besides iodine deficiency and excess many other dietary, environmental, and occupational factors are associated with the TNs incidence3,6-11. Among these factors a disturbance of evolutionary stable input of many trace elements (TEs) in human body after the industrial revolution plays a significant role in etiology of TNs12. Besides iodine, many other TEs have also essential physiological role and involved in thyroid functions13. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of TEs depend on tissue-specific need or tolerance, respectively13. Excessive accumulation or an imbalance of the TEs may disturb the cell functions and may result in cellular proliferation, degeneration, death, benign or malignant transformation13-15.

In our previous studies the complex of in vivo and in vitro nuclear analytical and related methods was developed and used for the investigation of iodine and other TEs contents in the normal and pathological thyroid16-22. Iodine level in the normal thyroid was investigated in relation to age, gender and some non-thyroidal diseases23,24. After that, variations of many TEs content with age in the thyroid of males and females were studied and age- and gender-dependence of some TEs was observed25-41. Furthermore, a significant difference between some TEs contents in colloid goiter, thyroiditis, thyroid adenoma, and cancer in comparison with normal thyroid and thyroid tissue adjacent to TNswas demonstrated42-48.

The aim of this study is to evaluate the possibilities of using differences in bromine (Br), copper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn) contents in nodular tissue, determined by a combination of non-destructive 109Cd (109Cd-EDXRF) and 241Am radionuclide-induced energy-dispersive X-ray fluorescent analysis (241Am-EDXRF), to diagnose thyroid malignancies and to evaluate the sensitivity, specificity, and accuracy for most informative TEs in the discrimination of TMN.

 

MATERIAL AND METHODS

 

Specimens and patients

 

Samples of the NT were obtained from randomly selected autopsy specimens of 105 deceased (European-Caucasian, mean age 44±21 years, range 2-87years), who had died suddenly. The majority of deaths were due to trauma. All the deceased had undergone routine autopsy at the Forensic Medicine Department of City Hospital, Obninsk. A histological examination in the NT group was used to control the age norm conformity, as well as to confirm the absence of micro-nodules and latent cancer. This examination was done in the Morbid Anatomy Department of City Hospital, Obninsk

All patients suffered from TBN (n=79, mean age M±SD was 44±11 years, range 22-64 years) and from TMN (n=41, mean age M±SD was 46±15 years, range 16-75years) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre (MRRC), Obninsk. Thick-needle puncture biopsy of suspicious nodules of the thyroid was performed for every patient, to permit morphological study of thyroid tissue at these sites and to estimate their TEs contents. In all cases the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. Histological conclusions for TBN were: 46 colloid goiter, 19 thyroid adenoma, 8 Hashimoto's thyroiditis, and 6 Riedel’s Struma, whereas for TMN were: 25 papillary adenocarcinomas, 8 follicular adenocarcinomas, 7 solid carcinomas, and 1 reticulosarcoma. Samples of nodular tissue for109 Cd-EDXRF and 241Am-EDXRF analysis was taken from both biopsy and resected materials.

Ethical approval

All studies were approved by the Ethical Committees of MRRC. All the procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments, or with comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Laboratory methods

All tissue samples obtained from NT, TBN and TMN were divided into two portions using a titanium scalpel to prevent contamination by TEs of stainless steel49. One was used for morphological study while the other was intended for TEs analysis. After the samples intended for TEs analysis were weighed, they were freeze-dried and homogenized50. 

To determine the contents of the TEs by comparison with known data for standard, aliquots of commercial, chemically pure compounds and synthetic reference materials were used51. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) were analyzed to estimate the precision and accuracy of results. The CRM IAEA H-4 subsamples were prepared in the same way like the samples of dry homogenized nodular tissue. Details of the relevant facility for 109Cd-EDXRF determination of Br, Cu, Fe, Rb, Sr, and Zn contents, methods of analysis and the quality control of results were presented in our earlier publications concerning the 109 Cd-EDXRF analysis of human thyroid and prostate tissue25,26,47,52. Detailed information on EDXRF determination of I contents with 241 Am radionuclide source, including methods of analysis and the quality control of results were presented in our earlier publication concerning the use of 241Am-EDXRF analysis in human thyroid study21.

Statistic

All samples for TEs analysis were prepared in duplicate and mean values of TEs contents were used in final calculation. Using Microsoft Office Excel software, some basic statistics, including, arithmetic mean, standard deviation of mean, standard error of mean, minimum and maximum values (range) was calculated for TEs contents in three groups of thyroid tissue (NT, TBN and TMN).The difference in the results between three groups of samples was evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

Sensitivity, specificity, and accuracy analysis

The possibility of “malignant or non- malignant” discrimination using results obtained in the study was estimated by such characteristics as “Sensitivity”, “Specificity”, and “Accuracy”. These characteristics were calculated.

 

RESULTS

 

Table 1 depicts certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, range) of the Br, Cu, Fe, I, Rb, Sr, and Zn mass fraction in thyroid tissue samples of three groups – NT, TBN and TMN. The ratios of means and the comparison of mean values of Br, Cu, Fe, I, Rb, Sr, and Zn mass fractions in pair of sample groups such as NT and TBN, NT and TMN, and also TBN and TMN is presented in Table 2. Figure 1 depicts individual data sets for Br, I, Rb, and Zn mass fraction in all samples of NT, TBN, and TMN group.

Parameters of the sensitivity, specificity and accuracy (M±95% confidence interval) of using I mass fraction for the diagnosis of thyroid malignancy are presented in Table 3. An estimation was made from comparison individual values in TMN group with those in NT and TBN groups combined. Value of I mass fraction equals 145 mg/kg dry tissue was chosen as upper limit (cut off) for thyroid malignancy.

The comparison of our results with published data (from 1990 year) for I mass fraction in NT27,28,31-34,37,53-72, TBN54,56,57,62,63,67-80, and TMN54,56,57,60,64-66,73,74,81-85 is shown in Table 4, Table 5, and Table 6, respectively. A number of values for TEs mass fractions were not expressed on a dry mass basis by the authors of the cited references. However, these values were calculated using published data for water (75%)86 and ash (4.16% on dry mass basis)87 contents in thyroid of adults. 

 

DISCUSSION

 

The results of the present study of the contents of Br, Cu, Fe, I, Rb, Sr and Zn in CRM IAEA H-4  samples analyzed by EDXRF are consistent with the previously reported results before21,25,26,47,52. It indicates acceptable accuracy of the TEs contents in NT, TBN, and TMN groups of tissue samples presented in Tables 1-3 and Figure 1. From Table 2, it is observed that in TMN tissue the mass fractions of I and Zn are significantly lower while the mass fraction of Rb is higher than in NT and TBN tissue. 

However, as illustrated in Figure 1, I content is the most informative parameter for the diagnosis of TMN (Fig. 1). When I level of 145 mg/kg dry tissue (M+SD) was chosen as the upper limit (cut off) for TMN tissue (Fig.1), results for a “malignant or non- malignant” determination from results obtained were the following: sensitivity 87±5%, specificity 96±2%, and accuracy 94±2%.The number of people examined was taken into account for calculation of confidence intervals88. In other words, if I contents in a nodule biopsy sample do not exceed 145 mg/kg dry tissue, one could diagnose a thyroid malignant tumor with an accuracy of 94±2%. Using the I-test makes it possible to diagnose thyroid malignancy in 87±5% cases (sensitivity).

Thus, I content in a nodule biopsy as biomarker of TMN could become a powerful diagnostic tool. To a large extent, the resumption of the search for new methods for diagnosis of TMN was due to experience gained in a critical assessment of the limited capacity of USS examination and cytological test of fine needle aspiration biopsy2-4. In addition to the USS examination and morphological study of needle-biopsy of the thyroid nodules, the I-test developed in the present study seems to be very useful. Experimental conditions of the present study were approximated to the hospital conditions as closely as possible. In all cases a part of the material obtained from a puncture needle biopsy of the affected site in the thyroid was analyzed. Therefore, our data allow us to evaluate adequately the importance of the I-test for the diagnosis of TMN. Obtained characteristics for accuracy, sensitivity, and specificity of the I-test 94%, 96%, and 87%, respectively, are significantly better than these parameters of the USS examination (nearly 80%)2,3. At that, the I-test gives a definite conclusion for all nodules investigated while using the morphological study of needle-biopsy up to 30% of nodules are categorized as cytologically “indeterminate”4.  Mean values obtained for I contents in NT, TBN, and TMN agree well with median of mean values published in scientific literature for the period from 1990 up to 2022 (Table 4 to Table 6).The range of I level means reported in the literature for NT, TBN, and TMN varies greatly (Table 4 to Table 6). This discrepancy can be explained by the dependence of the I content on many factors, including age, gender, race, body mass and stage of diseases, as not all of these factors were precisely controlled in previous studies. However, in our opinion, the main reasons for the inter-observer discrepancy can be attributed to the accuracy of analytical techniques and sample preparation methods and the inability to take standardized samples from affected tissues.

It was insufficient quality control of results in many previous studies. In some scientific reports, tissue samples were ashed or dried at high temperature for many hours. In other cases, thyroid samples were treated with solvents (distilled water, ethanol, formalin etc). There is evidence that during ashing, drying and digestion at high temperature some quantities of I are lost as a result of this treatment89-91.

It is well known that compared to other soft tissues, the human thyroid gland has significantly higher levels of I, because this element plays an important role in its normal functions, through the production of thyroid hormones (thyroxin and triiodothyronine) which are essential for cellular oxidation, growth, reproduction, and the activity of the central and autonomic nervous system. As was shown in present study, malignant transformation is accompanied by a significant loss of tissue-specific functional features, which leads to a drastically reduction in I content associated with functional characteristics of the human thyroid tissue. However, it is necessary to keep in mind that biochemical, or in other words, functional changes in thyroid cells are present from the earliest development of malignancy, which precedes any histopathological indication of malignancy, and these biochemical changes persist during progression of the malignancy and remain present in advanced thyroid cancer. Thus, I depletion is an early step in the malignant proliferation process and I depletion in nodular tissue precedes the morphological transformation of cells from being histopathologically benign to malignant12.

In our study the portable device was used for EDXRF analysis, with its 241Am source for the excitation of X-ray fluorescence in the needle biopsy sample, was developed by ourselves. More powerful devices for EDXRF analysis with X-ray tubes, including “the total reflection” version (TRXRF) of the method, allow reliable determinations of I and many other TEs contents in a microprobe of a human body tissues and fluids within a few minutes92. EDXRF is a fully instrumental and non-destructive method because sample is investigated without requiring any pretreatment or its consumption. Moreover, it is well known that among the most modern analytical technologies, EDXRF is one of the simplest, fastest, most reliable and efficient of the available techniques for TEs determination92. There are many different kinds of EDXRF and TRXRF device on the market and technical improvements are frequently announced. Thus, in our opinion, obtaining the I level in a needle biopsy of thyroid nodule, using EDXRF, is a fast, reliable and very informative diagnostic tool that can be successfully used as an additional test for diagnoses of thyroid malignancy.

 

CONCLUSION

 

We conclude from this study that EDXRF is a suitable analytical tool for the determination of the content of Br, Cu, Fe, I, Rb, Sr, and Zn in human thyroid tissue samples, including needle biopsy material. It was observed that in TMN tissue, the mean mass fractions for I and Zn were lower while the mean mass fraction for Rb was higher than in NT and TBN tissues. Also, the iodine nodular tissue content has been proven to be the most useful parameter for diagnosing malignant tumors of the thyroid gland. It was found that the 'sensitivity', 'specificity', and 'accuracy' of TMN determination using iodine level determination by needle biopsy of affected thyroid tissue were significantly higher than those using ultrasound examination and cytological testing of fine needle biopsy. Finally, we conclude that the study of iodine level in needle biopsy of TNs, obtained using EDXRF, is a fast, reliable and informative diagnostic tool that can be successfully used as an additional test to identify thyroid malignancy.

 

ACKNOWLEDGEMENTS

 

The authoris extremely grateful to Prof. B.M. Vtyurin and Prof. V.S. Medvedev, MRRC, Obninsk, as well as to Dr. Yu. Choporov, former Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples.

 

AUTHOR’S CONTRIBUTION

 

Vladimir Zaichick–idea and organization of the study, measurement of trace elements in thyroid tissue samples using EDXRF, insurance of the result accuracy, statistical analysis of results, review of literature data, writing of manuscript, translation on English.

 

CONFLICT OF INTEREST

 

No conflict of interest associated with this work.

 

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