Journal of Health Care and Research
ISSN: 2582-8967
Article Type: Original Article
DOI: 10.36502/2022/hcr.6207
J Health Care and Research. 2022 May 24;3(1):16-30
Diagnosis of Thyroid Malignancy using Levels of Chemical Element Contents in Nodular Tissue
Vladimir Zaichick1*
1Radionuclide Diagnostics Department, Medical Radiological Research Centre, Obninsk, Russia
Corresponding Author: Prof. Dr. Vladimir Zaichick, PhD, DSc, CChem, FRSC ORCID iD
Address: Medical Radiological Research Centre, Korolyev St.4, Obninsk 249036, Kaluga Region, Russia.
Received date: 14 March 2022; Accepted date: 16 May 2022; Published date: 24 May 2022
Citation: Zaichick V. Diagnosis of Thyroid Malignancy using Levels of Chemical Element Contents in Nodular Tissue. J Health Care and Research. 2022 May 24;3(1):16-30.
Copyright © 2022 Zaichick V. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited.
Keywords: Diagnosis of Thyroid Malignancy, Normal Thyroid, Thyroid Nodules, Chemical Elements, Neutron Activation Analysis, Inductively Coupled Plasma Atomic Emission Spectrometry
Abstract
Introduction: Thyroid benign (TBN) and malignant (TMN) 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 present study was to evaluate possibilities of using differences in chemical elements (ChEs) contents in nodular tissue for diagnosis of thyroid malignancy.
Methods: Contents of ChEs such as aluminum (Al), boron (B), barium (Ba), calcium (Ca), chlorine (Cl), coper (Cu), iron (Fe), iodine (I), potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), sulfur (S), silicon (Si), strontium (Sr), vanadium (V), 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 a combination of non-destructive and destructive methods: instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides and inductively coupled plasma atomic emission spectrometry.
Results: It was observed that in TMN tissue the mean mass fraction of I was lower while the mean mass fractions of K, Mg, and P were higher than in both NT and TBN groups of samples. It was demonstrated that I content 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 was significantly higher than that using US examination and cytological test of fine needle aspiration biopsy.
Conclusions: It was concluded that determination of the I level in a needle biopsy of TNs using non-destructive instrumental analytical method is a fast, reliable, and very informative diagnostic tool that can be successfully used as an additional test of thyroid malignancy identification.
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 TMN [2]. It is appropriate mention here that the incidence of TMN is increasing rapidly (about 5% each year) worldwide [2]. Surgical treatment is not always necessary for TBN whereas surgical treatment is required in TMN. Thus, differentiated TBN and TMN have a great influence on thyroid therapy.
Ultrasound (US) examination widely use as the primary method for early detection and diagnosis of the TNs. However, there are many similarities in the US characteristics of both TBN and TMN. For misdiagnosis prevention some computer-diagnosis systems based on the analysis of US images were developed, however as usual these systems for the diagnosis of TMN showed accuracy, sensitivity, and specificity nearly 80% [2,3]. Therefore, when US examination shows suspicious signs, an US-guided fine-needle aspiration biopsy is advised. Despite the fine needle aspiration biopsy has remained the diagnostic tool of choice for evaluation of US suspicious 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 shown that at the current time there is no perfect biochemical, immunological, and genetic biomarkers to discriminate malignancy [5]. Therefore, further improvements of TMN diagnostic accuracy are warranted.
During the last decades it was demonstrated that besides the iodine deficiency and excess many other dietary, environmental, and occupational factors are associated with the TNs incidence [3,6-11]. Among these factors a disturbance of evolutionary stable input of many chemical elements (ChEs) in human body after industrial revolution plays a significant role in etiology of TNs [12]. Besides iodine, many other ChEs have also essential physiological role and involved in thyroid functions [13]. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of ChEs depend on tissue-specific need or tolerance, respectively [13]. Excessive accumulation or an imbalance of the ChEs may disturb the cell functions and may result in cellular proliferation, degeneration, death, benign or malignant transformation [13-15].
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 ChEs contents in the normal and pathological thyroid [16-22]. Iodine level in the normal thyroid was investigated in relation to age, gender and some non-thyroidal diseases [23,24]. After that, variations of many ChEs content with age in the thyroid of males and females were studied and age- and gender-dependence of some ChEs was observed [25-41]. Furthermore, a significant difference between some ChEs contents in colloid goiter, thyroiditis, thyroid adenoma, and cancer in comparison with normal thyroid and thyroid tissue adjacent to TNs was demonstrated [42-48].
The present study had two aims. The main objective was to assess the aluminum (Al), boron (B), barium (Ba), calcium (Ca), chlorine (Cl), coper (Cu), iron (Fe), iodine (I), potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), sulfur (S), silicon (Si), strontium (Sr), vanadium (V), and zinc (Zn) contents in “normal” thyroid (NT) as well as in nodular tissue of patients who had either TBN or TMN using a combination of non-destructive instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR) and destructive method such as inductively coupled plasma atomic emission spectrometry (ICP-AES). The second aim was to evaluate ChEs content to aid diagnosis of thyroid malignancy.
Material and Methods
Samples of the NT were obtained from randomly selected autopsy specimens of 105 deceased (European-Caucasian, mean age 44±21 years, range 2-87), who had died suddenly. The majority of deaths were due to trauma. All the deceased were citizens of Obninsk and had undergone routine autopsy at the Forensic Medicine Department of City Hospital, Obninsk. A histological examination in the control group was used to control the age norm conformity, as well as to confirm the absence of micro-nodules and latent cancer.
All patients suffered from TBN (n=79, mean age M±SD was 44±11 years, range 22-64) and from TMN (n=41, mean age M±SD was 46±15 years, range 16-75) 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 goiters, 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 for ChEs analysis were taken from both biopsy and resected materials.
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.
All tissue samples obtained from NT, TBN and TMN were divided into two portions using a titanium scalpel to prevent contamination by ChEs of stainless steel [49]. One was used for morphological study while the other was intended for ChEs analysis. After the samples intended for TEs analysis were weighed, they were freeze-dried and homogenized [50].
To determine the contents of the ChEs by comparison with known data for standard, aliquots of commercial, chemically pure compounds and synthetic reference materials were used [51]. Ten sub-samples of certified reference material (CRM) IAEA H-4 (animal muscle) and five sub-samples of CRM of the Institute of Nuclear Chemistry and Technology (INCT, Warszawa, Poland) INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs were treated and analyzed in the same conditions that thyroid samples to estimate the precision and accuracy of results.
The pounded samples weighing about 10 mg (for biopsy) and 100 mg (for resected materials) were used for ChEs measurement by INAA-SLR. The content of Ca, Cl, I, K, Mg, Mn, and Na were determined by INAA-SLR using a horizontal channel equipped with the pneumatic rabbit system of the WWR-c research nuclear reactor (Branch of Karpov Institute, Obninsk). After non-destructive INAA-SLR investigation the thyroid samples were used for ICP-AES. The samples were decomposed in autoclaves and aliquots of solutions were used to determine the Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fractions by ICP-AES using the Spectrometer ICAP-61 (Thermo Jarrell Ash, USA). Information detailing with the NAA-SLR and ICP-AES methods used and other details of the analysis were presented in our earlier publications concerning ChEs contents in human thyroid [33,34] and prostate [52].
All samples for ChEs analysis were prepared in duplicate, and mean values of ChEs contents were used in final calculation. Mean values of ChEs contents were used in final calculation for the Ca, K, Mg, Mn, and Na mass fractions measured by two methods. 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 ChEs 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.
Results
Table-1 depicts certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, range) of the Al, B, Ba, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fraction in thyroid tissue samples of three groups – NT, TBN and TMN.
Table-1: Basic statistical parameters of Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fraction (mg/kg, dry mass basis) in normal thyroid (N) and in thyroid benign (TBN) and malignant (TMN) nodules
The ratios of means and the comparison of mean values of Al, B, Ba, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, 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.
Table-2: Ratio of means and the difference between mean values of Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fraction (mg/kg dry tissue) in normal thyroid (NT) and in thyroid benign (TBN) and malignant (TMN) nodules
Fig-1 depicts individual data sets for I, K, Mg, and P mass fraction in all samples of NT, TBN, and TMN group.
Fig-1
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, if value of I mass fraction equals 145 mg/kg dry tissue was chosen as upper limit (cut off) for thyroid malignancy.
Table-3: Parameters of the sensitivity, specificity and accuracy (M±95% confidence interval) of I mass fraction for the diagnosis of TMN (an estimation is made for “TMN or NT and TBN”)
The comparison of our results with published data (from 1990 year) for I mass fraction in NT [27,28,31-34,37,53-72], TBN [54,56,57,62,63,67-80], and TMN [54,56,57,60,64-66,73,74,81-85] is shown in Tables 4, 5, and 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, we calculated these values using published data for water (75%) [86] and ash (4.16% on dry mass basis) [87] contents in thyroid of adults.
Discussion
As was shown before [33,34,52] good agreement of the Al, B, Ba, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn contents in CRM IAEA H-4, INCT-SBF-4, INCT-TL-1, and INCT-MPH-2 samples determined by both INAA-SLR and ICP-AES methods with the certified data of these CRMs indicates acceptable accuracy of the results obtained in the study of NT, TBN, and TMN groups of tissue samples presented in Table-1, Table-2 and Table-3 and Fig-1.
From Table-2, it is observed that in TMN tissue the mass fractions of I is significantly lower while the mass fraction of K, Mg, and P is higher than in both NT and TBN groups of samples. However, as illustrated in Fig-1, I content is the most informative parameter for the diagnosis of TMN (Fig-1). If the I level of 145 mg/kg dry tissue (about M+SD for TMN group) is chosen as the upper limit (cut off) for TMN tissue (Fig-1), results for a “malignant or non- malignant” determination from results obtained would be the following:
Sensitivity = {correct positive test (CPT)/[CPT + false negative test (FNT)]}×100% = 84±6%;
Specificity = {correct negative test (CNT)/[CNT + false positive test (FPT)]} ×100% = 96±2%;
Accuracy = [(CPT+CNT)/(CPT+FNT+CNT+FPT)] ×100% = 94±2%.
The number of people examined was taken into account for calculation of confidence intervals [88]. In other words, if I contents in a nodule biopsy sample do not exceed 145 mg/kg dry tissue, one could diagnose a malignant tumor with an accuracy of 94±2%. Using the I-test makes it possible to diagnose thyroid malignancy in 84±6% 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 US examination and cytological test of fine needle aspiration biopsy [2-4]. In addition to the US 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 84, respectively, are significantly better than these parameters of the US 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 period from 1990 up to 2022 year (Table-4, Table-5, and Table-6, respectively). The range of means of I level reported in the literature for NT, TBN, and TMN vary widely (Table-4, Table-5, and Table-6). This can be explained by a dependence of I content on many factors, including age, gender, ethnicity, mass of the TNs, and the stage of diseases. Not all these factors were strictly controlled in cited studies. However, in our opinion, the leading causes of inter-observer variability can be attributed to the accuracy of the analytical techniques, sample preparation methods, and inability of taking uniform samples from the affected tissues. It was insufficient quality control of results in these studies. In many 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 treatment [89-91].
Table-4: Reference data of I mass fractions in “normal” human thyroid published from 1990 year
Table-5: Reference data of I mass fractions in thyroid benign nodules published from 1990 year
Table-6: Reference data of I mass fractions in thyroid malignant nodules published from 1990 year
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 malignant.
In our study non-destructive INAA-SLR was used for I determination. This method needs in using a nuclear reactor that is not always available in clinical practice. However, there is an alternative non-destructive method such as EDXRF analysis, including “the total reflection” version (TRXRF), which allows reliable determinations of I and many other ChEs contents in a microprobe of a human body tissues and fluids within a few minutes [92]. 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 ChEs determination [92]. 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
In this work, ChEs analysis was carried out in the tissue samples of NT and thyroid with TBN and TMN using a combination of non-destructive INAA-SLR and destructive ICP-AES methods. It was shown that the combination of these two methods is an adequate analytical tool for the non-destructive determination of nineteen ChEs (Al, B, Ba, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn) contents in the tissue samples of human thyroid, including needle-biopsy material. It was observed that in TMN tissue the mean mass fraction of I was lower while the mean mass fractions of K, Mg, and P were higher than in both NT and TBN groups of samples. It was demonstrated that I content 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 was significantly higher than that using US examination and cytological test of fine needle aspiration biopsy. It was concluded that determination of the I level in a needle biopsy of TNs, using non-destructive instrumental analytical method, is a fast, reliable, and very informative diagnostic tool that can be successfully used as an additional test of thyroid malignancy identification.
Acknowledgements
The author is extremely grateful to Profs. Vtyurin BM and Medvedev VS, Medical Radiological Research Center, Obninsk, as well as to Dr. Choporov Yu, former Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples. The author is also grateful to Dr. Karandaschev V, Dr. Nosenko S, and Moskvina I, Institute of Microelectronics Technology and High Purity Materials, Chernogolovka, Russia, for their help in ICP-AES analysis.
Funding
There were no any sources of funding that have supported this work.
Conflict of Interest
The author has not declared any conflict of interests.
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