
==== Front
Rom J Morphol Embryol
Rom J Morphol Embryol
RJME
Romanian Journal of Morphology and Embryology
1220-0522
2066-8279
Academy of Medical Sciences, Romanian Academy Publishing House, Bucharest

39020541
650224267271
10.47162/RJME.65.2.13
Original Paper
The expression of c-MYC, Cyclin D1 and Ki-67/MIB-1 in benign and malignant thyroid tissues: is there any diagnostic value?
Forozidou Evropi 1
Syrnioti Antonia 2
Laskou Styliani 3
Poutoglidis Alexandros 4
Sapalidis Konstantinos 3
Koletsa Triantafyllia 2
1 Department of Otorhinolaryngology, Head and Neck Surgery, George Papanikolaou General Hospital, Thessaloniki, Greece
2 Department of Pathology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece
3 3rd Surgical Department, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece
4 Department of Anatomy and Surgical Anatomy, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece
Corresponding Author: Alexandros Poutoglidis, MD, PhD Department of Anatomy and Surgical Anatomy, School of Medicine, Faculty of Health Sciences Aristotle University of Thessaloniki AUTH Campus 54124 TK, Thessaloniki Greece + 30 2310 999 900 xilouris21@gmail.com
Apr-Jun 2024
30 6 2024
65 2 267271
21 12 2023
26 5 2024
Copyright © 2024, Academy of Medical Sciences, Romanian Academy Publishing House, Bucharest
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open-access article distributed under the terms of a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License, which permits unrestricted use, adaptation, distribution and reproduction in any medium, non-commercially, provided the new creations are licensed under identical terms as the original work and the original work is properly cited.
Aim: To investigate the immunohistochemical (IHC) expression and the diagnostic value of c-MYC, Cyclin D1, and Ki-67/MIB-1 in follicular adenomas (FAs), follicular carcinomas (FCs), and anaplastic carcinomas (ACs) of the thyroid gland, as well as in their corresponding adjacent, non-neoplastic thyroid tissue (NNTT). Materials and Methods: We conducted a retrospective study of patients who were pathologically diagnosed with FA, FC, or AC after total thyroidectomy. Tissue microarrays with cores taken from neoplastic and adjacent NNTT were constructed. Immunohistochemistry for anti-c-MYC, anti-Cyclin D1, and anti-Ki-67/MIB-1 antibodies was performed, and the positivity was evaluated. Results: Twenty-eight specimens were included. Nuclear c-MYC positivity was observed in 4/11 FCs, and 3/4 ACs, whereas cytoplasmic c-MYC positivity was found in 16/24 NNTTs. Globally, there were statistically significant differences between neoplasms and NNTTs, with higher nuclear c-MYC and Cyclin D1 expression observed in neoplasms (p=0.017 and p=0.001, respectively). In contrast, cytoplasmic positivity was seen solely in NNTTs (p=0.001). Cyclin D1 positivity was noted in 11/13 FAs, 7/11 FCs, 2/4 ATCs, and only in one NNTT. A statistically significant correlation was found between MIB1 and c-MYC nuclear positivity (p=0.040). Conclusions: Our findings exhibit a clear difference in the IHC expression of c-MYC and Cyclin D1 between different types of thyroid tumors, as well as between the neoplastic and NNTT. Nuclear c-MYC positivity excludes the benign nature of a thyroid lesion, in contrast to cytoplasmic positivity, which demonstrates normal or hyperplastic nature.

c-MYC
Cyclin D1
Ki-67/MIB-1
molecular markers
follicular adenomas
thyroid cancer
==== Body
pmcIntroduction

Thyroid cancer is the most common malignancy of the endocrine system [1]. Papillary carcinomas (PCs) and follicular carcinomas (FCs) are mostly associated with favorable prognosis. On the other hand, medullary carcinomas (MCs) and especially anaplastic carcinomas (ACs) are considered entities with much worse outcome [2].

The molecular genetic alterations leading to carcinogenesis are not well understood. The presence of specific oncogenes or mutations to tumor suppressor genes has been related to the progression of well-differentiated neoplasms to poorly differentiated tumors [3].

c-MYC is a proto-oncogene encoding a nuclear phosphoprotein responsible for tumor growth [4]. Both cytoplasmic and nuclear overexpression of c-MYC have been linked to tumorigenesis. The role of c-MYC in thyroid cancer has been investigated in limited studies with controversial results [5, 6].

Cyclin D1 acts as a regulator of cyclin-dependent kinase (CDK)4 and CDK6, having a crucial role in the cell cycle progression [7]. According to literature, Cyclin D1 promotes tumor progression in renal cancer, gastric cancer, and lung cancer [8, 9, 10]. There is also a report about the high levels of its expression in thyroid cancer [11]. Recently, increased expression of Cyclin D1 and c-MYC has been associated with aggressive clinical behavior of papillary thyroid carcinoma. Enhanced expression of Cyclin D1 and c-MYC seems to be a prognostic factor and possible mechanism for recurrence of papillary thyroid carcinoma [12]. However, there is limited data for follicular adenomas (FAs) and carcinomas.

Ki-67/MIB-1 proliferation rate is used to determine the clinical behavior of thyroid gland tumors. Normal follicular cells of the thyroid gland have a proliferation rate of <0.1–0.3% [13]. In cases of carcinomas, the Ki-67 proliferation rate increases reversely proportionally to the cellular differentiation of the tumor with ACs having a proliferation rate of >30%. The expression of Ki-67/MIB-1 is related proportionally with the aggressiveness of thyroid cancer [14]. A deeper understanding of thyroid cancer genetics will possibly have diagnostic and therapeutic value.

Aim

The aim of our study was to investigate the immunohistochemical (IHC) expression patterns of c-MYC, Cyclin D1, and Ki-67/MIB-1 in FAs, FCs, and ACs of the thyroid gland, and the potential use of these markers for diagnostic purposes.

Materials and Methods

A systematic retrospective review was performed on all patients that underwent a total thyroidectomy in the 3rd Surgical Department of Aristotle University of Thessaloniki, AHEPA University Hospital, Thessaloniki, Greece, during the last five years. Patients who were pathologically diagnosed with FA, FC or AC were eligible for inclusion in our study. Every other patient was excluded. The medical charts of eligible patients were retrieved and the demographic data, preoperative hormonological status, and the presence of a known pathology of a gland (i.e., goiter) were recorded. Gross pathology characteristics of thyroid specimens, i.e., macroscopic nodules/lesions, their localization, and dimensions, were recorded from the histopathological report.

The respective eligible formalin-fixed paraffin-embedded tissue specimens were retrieved from the Department of Pathology of Aristotle University of Thessaloniki.

Briefly, we conducted a microscopic evaluation of Hematoxylin–Eosin (HE)-stained sections to confirm the previous pathological diagnosis according to the current classification of thyroid tumors. Selected areas, representative of the whole lesion, were marked for the construction of the tissue microarrays (TMAs). TMAs were constructed by using two cores from the neoplastic and one core from the adjacent non-neoplastic tissue (NNT) for each case. In addition, several other tissues were used at the edges of TMAs for orientation and control purposes. Three μm unstained sections from TMAs blocks were cut for HE and immunostains. IHC method was performed by using anti-c-MYC (rabbit monoclonal, clone EP121, titer 1/30, Zeta Corporation), anti-Cyclin D1 (rabbit monoclonal, clone EP12, titer 1/30, Dako), and anti-Ki-67/MIB-1 (mouse monoclonal, clone NCL-L-Ki67 MM1, titer 1/70, Leica Biosystems) antibodies.

A semiquantitative evaluation of each protein expression in every specimen was conducted. The cut-off level for considering negative expression was less than 5%.

A Microsoft Excel sheet was employed to record the data of the included specimens. The statistical analysis was made by the IBM Statistical Package for Social Sciences (SPSS) Statistics v25 program. The association between the immunohistochemistry parameters and the nature of the neoplasm was conducted with Fisher’s exact test to enhance the accuracy of the results.

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments and was approved by the Ethical and Research Committee of the Aristotle University of Thessaloniki (Approval No. 103/2023).

Results

The non-parametric Kruskal–Wallis’ test was employed to evaluate the difference among the four quality variables and, more specifically, the cytoplasmic c-MYC (c-MYC cyto %), the nuclear c-MYC (c-MYC nuclear %), the nuclear Cyclin D1 (Cyclin D1 nuclear %), and Ki-67/MIB-1 expression in the selected neoplasms. This test was conducted to overcome the lack of normal distribution of our specimens due to the limited population of our study. As for the comparison between the neoplastic and adjacent NNT, the non-parametric Wilcoxon signed-rank test was used to investigate the presence of statistically significant results.

The overall population of our study was 28 specimens, of which only five (18%) were male, confirming the increased incidence of thyroid neoplasms in the female population. From the histopathology reports, 13 (46.4%) specimens were diagnosed as FA, 11 (39.2%) specimens as FC, whereas ACs were only four (14.4%). The age of the patients ranged from 27–86 years with a median age of 52.85 years [standard deviation (SD): 15.20 years]. The highest dimension of the tumors had a median of 3.2 cm (SD: 2.01 cm). As for the location, 60.7% of the lesions were identified in the right lobe of the thyroid gland and 53.6% in the upper pole. In 21.4% of the lesions, the tumor occupied entirely the lobe of the gland. The histopathological types and localization of the neoplasms are illustrated in Table 1.

Table 1 Histopathological types and localization of the neoplasms

Type of neoplasm

	n (%)

	
FA

	13 (46.4%)

	
FC

	11 (39.2%)

	
AC

	4 (14.4%)

	
Position of the lesion

		
Right lobe

	17 (60.7%)

	
Left lobe

	9 (32.1%)

	
Isthmus

	2 (7.2%)

	
Location of the lesion

		
Upper pole

	15 (53.6%)

	
Lower pole

	5 (17.8%)

	
Isthmus

	2 (7.2%)

	
Total lobe

	6 (21.4%)

	
AC: Anaplastic carcinoma; FA: Follicular adenoma; FC: Follicular carcinoma; n: No. of cases

All four ACs completely conquered the involved lobe, and in one case infiltration of the striated muscles and perichondrium of the trachea was noticed. Preoperatively, 68% of patients were euthyroid (independently of their pathology), while only two (7%) of them were presented with hyperthyroidism. Both of the latter were diagnosed with FC. In regard to IHC results, nuclear positivity to anti-c-MYC antibody was recorded in four out of 11 FCs and in three out of four ACs, while cytoplasmic positivity in c-MYC was identified in 16 out of 24 NNTs (Figure 1A, 1B, 1C, 1D). According to the Kruskal–Wallis’ test, the c-MYC nuclear % and Ki-67/MIB-1 markers demonstrated statistically significant lower levels of expression in the adenomas compared to the AC and FC (p<0.001, p=0.004). The Ki-67/MIB-1 proliferative index was expressed in a percentage above 5% in three out of 11 FCs, and in none of FAs.

Statistically significant differences were recorded in the expression of markers between the neoplastic and the adjacent NNTs with higher nuclear positivity of c-MYC and Cyclin D1 in the neoplastic tissue (p=0.017 and p=0.001, respectively). On the other hand, cytoplasmatic granular positivity of c-MYC was recorded exclusively in the NNT (p=0.001). Cyclin D1 positivity was demonstrated in 11 out of 13 FAs, in seven out of 11 FCs, in two out of four ACs, and only in one NNT with weak immunoreactivity. Positivity for anti-Ki-67/MIB-1 antibody was recorded in three out of 11 FCs and in three out of four ACs, while it showed positivity in <5% in FAs.

As for the combined assessment of the various markers, there was found a statistically significant association between Ki-67/MIB-1 and c-MYC nuclear expression (r=0.413, p=0.040) (Table 2).

Table 2 Associations between the studied markers

Associations between markers

	Cyclin D1 nuclear (%)

	Ki-67/MIB-1 (%)

	
c-MYC nuclear (%)

	Correlation coefficient

	-0.088

	0.413

		
Sig. (2-tailed)

	0.670

	0.040

		
( N )

	26

	25

		
Cyclin D1 nuclear (%)

	Correlation coefficient

		-0.025

		
Sig. (2-tailed)

		0.907

		
( N )

		25

		

Figure 1 c-MYC immunohistochemical patterns in thyroid neoplasms and NNT: nuclear positivity in FC (a) and ATC (b), negativity in FA (c) of the thyroid gland, and cytoplasmic granular positivity in NNT (d). ATC: Anaplastic thyroid carcinoma; FA: Follicular adenoma; FC: Follicular carcinoma; NNT: Non-neoplastic tissue. Scale bar: (a, b and d) 50 μm; (c) 100 μm

Discussions

Genetics may determine the biological behavior of thyroid tumors. Some studies associated specific oncogenes and tumor suppressor genes with the transformation of well-differentiated thyroid neoplasms to poorly differentiated [15]. However, other studies failed to confirm this hypothesis [4, 5, 6].

Studies investigating the expression of c-MYC, Cyclin D1, and Ki-67/MIB-1 proteins in thyroid cancer are limited with controversial results. Specifically, the nuclear and cytoplasmic positivity of c-MYC is related with oncogenesis [16, 17]. There is no agreement for the presence and the level of c-MYC protein expression in neoplastic and NNTs [18, 19]. In this line, some previous studies failed to demonstrate nuclear positivity of c-MYC in thyroid tumors [20, 21]. Of note, the majority of these studies consisted of limited population and were based on the evaluation of IHC expression of c-MYC protein, mainly in papillary thyroid carcinomas [22]. There are no previous studies investigating c-MYC expression if FAs and carcinomas. In our study, the nuclear expression of c-MYC was recorded exclusively in carcinomas and not in adenomas or in the adjacent NNT. Therefore, nuclear positivity of c-MYC may be employed as a potential marker to differentiate the FAs from FCs, especially when histological features were ambiguous for the nature of a neoplasm. As for the cytoplasmic expression of c-MYC, a significant expression in the adjacent NNT was recorded, whereas no cytoplasmic immunostaining localization was evident in neoplastic thyroid tissues. Cyclin D1 promotes the transition of the cellular cycle from phase G1 to phase S [23]. Studies related the expression of this protein in a variety of thyroid neoplasms [12, 24]. Cyclin D1 expression presents heterogeneity among thyroid neoplasms and an almost similar positivity between FAs and FCs has been recorded [25, 26]. The majority of studies refer to different levels of Cyclin D1 expression in adenomas; however, other studies support the absence of expression [27, 28, 29]. These controversial results may be explained by the different methods and classification systems being recruited among studies. Our results demonstrate expression of Cyclin D1 in both FAs and FCs, but with variability on percentage and intensity expression. On the other hand, no Cyclin D1 expression was observed in the adjacent NNT.

The lack of balance between cellular proliferation and cellular apoptosis is considered the predominant pathway of tumorigenesis. The proliferation rate of malignant cells determines the biological behavior of tumors. The increased expression of Ki-67/MIB-1 (the most representative proliferation marker) is associated with increased tumor invasiveness and worse prognosis in various types of cancers. In thyroid cancer, Ki-67/MIB-1 expression is related to the progression of the tumor and the presence of distant or regional metastasis. However, the role of this marker in diagnosis of thyroid cancer is doubtful [14]. In our study, the expression of Ki-67/MIB-1 was not evaluated as statistically significant between carcinomas and adenomas. In other studies, the combination of Ki-67/MIB-1 with other markers has been employed to correlate the prognosis of FCs [30, 31].

We combined the expression of Ki-67/MIB-1 marker with the nuclear c-MYC and Cyclin D1 in various thyroid neoplasms, which showed statistical significance between Ki-67/MIB-1 and c-MYC nuclear expression, suggesting that the combination of these two markers may offer greater diagnostic value in distinguishing a carcinoma. A combination of Ki-67/MIB-1 and Cyclin D1 did not present any diagnostic significance.

A limitation of the present study is the small sample of tissues studied.

Conclusions

In the present study, nuclear positivity of c-MYC protein was demonstrated with a statistically significant difference in FCs and ACs compared to adenomas and NNT, thus speculating its implication in thyroid carcinogenesis. Specifically, nuclear c-MYC positivity excludes the benign nature of a thyroid lesion, in contrast to cytoplasmic positivity, which supports the normal or hyperplastic nature. Although a correlation between MIB-1 and c-MYC was observed, the diagnostic value of c-MYC localization proves to be superior. Due to the lack of literature, it becomes necessary to design prospective studies to further investigate the combination of IHC markers that will allow the improvement of the diagnosis of thyroid neoplasms, in particular of those with ambiguous histological features.

Conflict of interests

The authors declare that they have no conflict of interests.
==== Refs
References

1 Cabanillas ME McFadden DG Durante C Thyroid cancer Lancet 2016 388 10061 2783 2795 27240885
2 Nabhan F Dedhia PH Ringel MD Thyroid cancer, recent advances in diagnosis and therapy Int J Cancer 2021 149 5 984 992 34013533
3 Sakr HI Chute DJ Nasr C Sturgis CD cMYC expression in thyroid follicular cell-derived carcinomas: a role in thyroid tumorigenesis Diagn Pathol 2017 12 1 71 71 28974238
4 Wiseman SM Masoudi H Niblock P Turbin D Rajput A Hay J Bugis S Filipenko D Huntsman D Gilks B Anaplastic thyroid carcinoma: expression profile of targets for therapy offers new insights for disease treatment Ann Surg Oncol 2007 14 2 719 729 17115102
5 Soares P Lima J Preto A Castro P Vinagre J Celestino R Couto JP Prazeres H Eloy C Máximo V Sobrinho-Simões M Genetic alterations in poorly differentiated and undifferentiated thyroid carcinomas Curr Genomics 2011 12 8 609 617 22654560
6 Smith N Nucera C Personalized therapy in patients with anaplastic thyroid cancer: targeting genetic and epigenetic alterations J Clin Endocrinol Metab 2015 100 1 35 42 25347569
7 Li XJ Wen R Wen DY Lin P Pan DH Zhang LJ He Y Shi L Qin YY Lai YH Lai JN Yang JL Lai QQ Wang J Ma J Yang H Pang YY Downregulation of miR-193a-3p via targeting cyclin D1 in thyroid cancer Mol Med Rep 2020 22 3 2199 2218 32705210
8 Zhao M Xu P Liu Z Zhen Y Chen Y Liu Y Fu Q Deng X Liang Z Li Y Lin X Fang W Dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal and PTEN-suppressing Wnt/β-catenin signaling in non-small-cell lung cancer Cell Death Dis 2018 9 2 78 78 29362431
9 Huang H Han Y Yang X Li M Zhu R Hu J Zhang X Wei R Li K Gao R HNRNPK inhibits gastric cancer cell proliferation through p53/p21/CCND1 pathway Oncotarget 2017 8 61 103364 103374 29262567
10 Xue J Qin Z Li X Zhang J Zheng Y Xu W Cao Q Wang Z Genetic polymorphisms in cyclin D1 are associated with risk of renal cell cancer in the Chinese population Oncotarget 2017 8 46 80889 80899 29113352
11 Cheng S Serra S Mercado M Ezzat S Asa SL A high-throughput proteomic approach provides distinct signatures for thyroid cancer behavior Clin Cancer Res 2011 17 8 2385 2394 21389096
12 Sanjari M Kordestani Z Safavi M Mashrouteh M FekriSoofiAbadi M Tafreshi AG Enhanced expression of Cyclin D1 and C-myc, a prognostic factor and possible mechanism for recurrence of papillary thyroid carcinoma Sci Rep 2020 10 1 5100 5100 32198408
13 Saad AG Kumar S Ron E Lubin JH Stanek J Bove KE Nikiforov YE Proliferative activity of human thyroid cells in various age groups and its correlation with the risk of thyroid cancer after radiation exposure J Clin Endocrinol Metab 2006 91 7 2672 2677 16670159
14 Pan DH Wen DY Luo YH Chen G Yang H Chen JQ He Y The diagnostic and prognostic values of Ki-67/MIB-1 expression in thyroid cancer: a meta-analysis with 6,051 cases Onco Targets Ther 2017 2017 10 3261 3276
15 Saltman B Singh B Hedvat CV Wreesmann VB Ghossein R Patterns of expression of cell cycle/apoptosis genes along the spectrum of thyroid carcinoma progression Surgery 2006 140 6 899 905 17188136
16 Santoro M Papotti M Chiappetta G Garcia-Rostan G Volante M Johnson C Camp RL Pentimalli F Monaco C Herrero A Carcangiu ML Fusco A Tallini G RET activation and clinicopathologic features in poorly differentiated thyroid tumors J Clin Endocrinol Metab 2002 87 1 370 379 11788678
17 Puxeddu E Moretti S Elisei R Romei C Pascucci R Martinelli M Marino C Avenia N Rossi ED Fadda G Cavaliere A Ribacchi R Falorni A Pontecorvi A Pacini F Pinchera A Santeusanio F BRAFV599E mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas J Clin Endocrinol Metab 2004 89 5 2414 2420 15126572
18 Masood S Auguste LJ Westerband A Belluco C Valderama E Attie J Differential oncogenic expression in thyroid follicular and Hürthle cell carcinomas Am J Surg 1993 166 4 366 368 8214294
19 Braunschweig T Kaserer K Chung JY Bilke S Krizman D Knezevic V Hewitt SM Proteomic expression profiling of thyroid neoplasms Proteomics Clin Appl 2007 1 3 264 271 21136677
20 Bai MK Costopoulos JS Christoforidou BP Papadimitriou CS Immunohistochemical detection of the c-myc oncogene product in normal, hyperplastic and carcinomatous endometrium Oncology 1994 51 4 314 319 8208512
21 Bircan S Ensari A Ozturk S Erdogan N Dundar I Ortac F Immunohistochemical analysis of c-myc, c-jun and estrogen receptor in normal, hyperplastic and neoplastic endometrium Pathol Oncol Res 2005 11 1 32 39 15800680
22 Mizukami Y Nonomura A Hashimoto T Michigishi T Noguchi M Matsubara F Yanaihara N Immunohistochemical demonstration of epidermal growth factor and c-myc oncogene product in normal, benign and malignant thyroid tissues Histopathology 1991 18 1 11 18 2013457
23 Hashimoto T Matsubara F Mizukami Y Miyazaki I Michigishi T Yanaihara N Tumor markers and oncogene expression in thyroid cancer using biochemical and immunohistochemical studies Endocrinol Jpn 1990 37 2 247 254 1699752
24 Helin K Regulation of cell proliferation by the E2F transcription factors Curr Opin Genet Dev 1998 8 1 28 35 9529602
25 Erickson LA Jin L Goellner JR Lohse C Pankratz VS Zukerberg LR Thompson GB van Heerden Grant CS Lloyd RV Pathologic features, proliferative activity, and cyclin D1 expression in Hurthle cell neoplasms of the thyroid Mod Pathol 2000 13 2 186 192 10697277
26 Wang S Wuu J Savas L Patwardhan N Khan A The role of cell cycle regulatory proteins, cyclin D1, cyclin E, and p27 in thyroid carcinogenesis Hum Pathol 1998 29 11 1304 1309 9824112
27 Wang S Lloyd RV Hutzler MJ Safran MS Patwardhan NA Khan A The role of cell cycle regulatory protein, cyclin D1, in the progression of thyroid cancer Mod Pathol 2000 13 8 882 887 10955455
28 Zou M Shi Y Farid NR Al-Sedairy ST Inverse association between cyclin D1 overexpression and retinoblastoma gene mutation in thyroid carcinomas Endocrine 1998 8 1 61 64 9666346
29 Lazzereschi D Sambuco L Carnovale Scalzo Ranieri A Mincione G Nardi F Colletta G Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas Int J Cancer 1998 76 6 806 811 9626345
30 Tang J Gui C Qiu S Wang M The clinicopathological significance of Ki67 in papillary thyroid carcinoma: a suitable indicator World J Surg Oncol 2018 16 1 100 100 29855303
31 Matsuse M Yabuta T Saenko V Hirokawa M Nishihara E Suzuki K Yamashita S Miyauchi A Mitsutake N TERT promoter mutations and Ki-67 labeling index as a prognostic marker of papillary thyroid carcinomas: combination of two independent factors Sci Rep 2017 7 41752 41752 28150740
