TSHR expression and methylation level changes as diagnostic and prognostic biomarker

1

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

FACULTY OF MEDICINE

Department of Endocrinology

Matan Klein

TSHR expression and methylation level changes as diagnostic and

prognostic biomarker

in papillary thyroid cancer: a systematic review

MASTER THESES

Of Integrated Studies of Medicine

2

TABLE OF CONTENTS

SUMMARY (ABSTRACT) ... 3

ACKNOWLEDGMENTS... 4

CONFLICT OF INTEREST ... 4

ETHICS COMMITTEE APPROVAL ... 4

ABBREVIATIONS LIST ... 5

TERMS ... 6

INTRODUCTION... 7

AIM AND OBJECTIVES OF THE THESIS... 9

RESEARCH METHODOLOGY AND METHODS ... 10

Material and Methods ... 10

Search Question: ... 10

Search Strategy: ... 10

Screening ... 11

Eligibility ... 12

Studies included in analysis ... 13

Risk of Bias ... 13

SYSTEMIZATION AND ANALYSIS OF DATA ... 14

ANALYSIS OF DATA ... 15

DISCUSSION ... 24

CONCLUSION/RECOMMENDATIONS ... 26

REFERENCES ... 28

3

SUMMARY (ABSTRACT)

Title: TSHR expression and methylation level changes as diagnostic and prognostic biomarker in papillary thyroid cancer: a systematic review.

Aims and Objectives: the aim of study is to systematically assess studies evaluating TSH receptor (TSHR) expression and methylation level changes as diagnostic and prognostic biomarkers in papillary thyroid cancer. Objectives of the study include: 1. Systematically group and qualitatively assess studies on TSHR expression level changes as a diagnostic marker for papillary thyroid cancer. 2. Systematically group and qualitatively assess TSHR expression level changes as a prognostic marker evaluation in papillary thyroid cancer studies. 3. To review the potential value of correlation analysis between methylation status of TSHR gene and TSHR expression level changes in diagnosis and prognosis in papillary thyroid cancer patients.

Methodology: After a search on NCBI PubMed, this review assessed total of 264 articles with specific inclusion and exclusion criteria. Overall, 28 articles were included. Studies further divided into two main groups: one group for analysis of early diagnosis and prognostic value (disease recurrence and non-recurrence) by TSHR expression level changes in PTC patients and another group for analysing the potential significance of TSHR gene methylation level changes in PTC. The risk of bias of the individual studies evaluated using a modified Newcastle-Ottawa Scale (NOS) for observational studies.

Results: this study reviewed 28 studies exploring the early diagnostic and prognostic value of TSHR expression level changes in PTC. 19 studies of TSHR mRNA and 9 studies of TSHR gene methylation status had statistically significant association with early diagnosis and prognostic (recurrence) prediction in PTC patients. 15 studies with total sample size of 1,058 supported the hypothesis that TSHR expression level changes can be independent early diagnosis marker in PTC. As a prognostic marker, high TSHR expression level may be used in prediction of PTC recurrence, local lymph node metastasis, distant metastasis and a higher TNM stage. In 9 studies with total sample size of 782 were found that TSHR hypermethylation lead to downregulation (low expression), repression and silence of TSHR gene that may be associated with PTC carcinogenesis.

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ACKNOWLEDGMENTS

I would like to express my deepest appreciation to Prof. Birutė Žilaitienė for her patience, guidance, and encouragement throughout all the process of my master thesis.

Additionally, I would like to thank to Mintaute Kazokaite and Dr. Raimonda Klimaite from Endocrinology Department of Lithuanian University of Health Sciences and everyone who make this work possible.

CONFLICT OF INTEREST

The author declare no conflicts of interest.

ETHICS COMMITTEE APPROVAL

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ABBREVIATIONS LIST

Thyroid stimulating hormone receptor Papillary thyroid carcinoma

Classic papillary thyroid carcinoma Papillary thyroid micro carcinoma Thyroid carcinoma

Poorly differentiated cancer Undifferentiated carcinoma Healthy control

Messenger RNA

Methylation specific polymerase chain reaction. Chromatin immune histochemical analysis Combined bisulphite restriction analysis Fine needle aspiration cytology

Preferred reporting items for systematic reviews and meta-analysis. Ultrasound

Computed tomography

Positron emission tomography

Vs Versus

PCR Polymerase chain reaction

qRT-PCR Quantitative reverse transcription polymerase chain reaction

TNM Tumour, nodule, metastasis staging

L.N Lymph node

LNM Lymph node metastasis

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TERMS

TSHR expression level - TSHR expression level is detected by using quantitative mRNA detection methods that allow determining high expression level or low expression level of TSHR.

TSHR gene methylation - natural epigenetic modification of DNA, consists of covalent addition of methyl group to the five carbon of the cytosine. Methylation is a key player in regulation of gene expression. Methylation can change the activity of DNA segment without changing the sequence. Methylation of cytosine in the promoter region of TSHR gene inhibits transcriptional binding and hence gene expression. Evidence points to complex molecular events that activates proto-oncogenes, and silences tumour suppressor genes, leading to the development of cancer.

Hypermethylation - biological process by which methyl groups are added to the CpG islands in DNA molecule. Gene promoter hypermethylation typically repress gene transcription. Silencing of gene expression, functionally important in neoplastic process, affect all cellular pathways, may cause TSHR down regulation and TSHR low expression.

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INTRODUCTION

There are several histological types and subtypes of thyroid cancer with different cellular origins, the 4 major subtype of thyroid carcinoma are: papillary (80%), follicular (15%), anaplastic (5%) and medullary (5%). Most carcinoma derived from the follicular epithelium of the gland and the majority of them are well-differentiated lesions. Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy and the most common type of thyroid cancer representing around 80%[1,2]. The incidence of thyroid cancer has risen significantly over the past 4 decades and has emerged as a major health issue[3]. Although PTC usually have favourable prognosis with 10 years survival rate of more than 90% patients, some studies presented relative high recurrence after treatment around 20%, this associated with poor prognosis, increased incurability and patient mortality[4]. Follow up relies on combination of several methods: monitoring serum thyroglobulin (Tg), radioactive iodine whole body scans (WBS) and neck ultrasound. Each method has limitations that impair their accuracy as independent diagnostic test[5].

Ultrasound guided thyroid fine needle aspiration biopsy (FNAB) is the most successful biopsy method to diagnose thyroid cancer. For PTC, FNAB has proven high accurate. However, 15%-40% of FNA specimens have, undetermined cytology features that lead surgery for definitive diagnosis. Some of them will be thyroid carcinoma but most will be benign in final pathology. A more accurate pre-operative early diagnostic method could stratify patients to receive the appropriate treatment of surgery in case of malignancy and potentially avoid surgery as a diagnostic measure for those with benign conditions.

Moreover, effective follow-up of thyroid cancer patients can be a problem in some patients. Thyroid cancer may display features that impair Tg production, radioactive iodine uptake or both. Another 15-25% of patients have antibodies that interfere with Tg measurement. Furthermore, the immunoassay used for detecting Tg has low sensitivity during thyroid stimulating hormone suppression therapy. Alternative imaging tests with higher costs and variable accuracy, such as PET or CT scans, have a limited role in resolving these diagnostic and prognostic problems. These challenges illustrate the need for potential early diagnostic and prognostic biomarker for thyroid cancer.

8 Whose main functions are to regulate and control the growth also differentiation of thyroid cells. In recent years studies was found that primary papillary thyroid carcinoma (PTC) metastasis is accompanied with TSHR expression[8,9].

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AIM AND OBJECTIVES OF THE THESIS

To systematically assess studies evaluating TSH receptor (TSHR) expression and methylation level changes as diagnostic and prognostic biomarkers in papillary thyroid cancer.

Objectives:

1. Systematically group and qualitatively assess studies on TSHR expression level changes as a diagnostic marker for papillary thyroid cancer

2. Systematically group and qualitatively assess TSHR expression level changes as a prognostic marker evaluation in papillary thyroid cancer studies.

3. To review the potential value of correlation analysis between methylation status of TSHR gene and TSHR expression level changes in diagnosis and prognosis in papillary thyroid cancer patients.

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RESEARCH METHODOLOGY AND METHODS

Material and Methods

The presented review was conducted during the 2018 up to March 2020. Finished in the last year of Medicine studies in Lithuanian University of Health Sciences (LSMU). The protocol for the review was designed in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA)[10] which is an evidence-based set of items and serves as a protocol on the reporting of systematics reviews and meta-analyses. A detailed criteria checklist and a specifically designed outflow chart as seen in Figure 1 was used.

Research question

The research question formed according to the PICO (population, intervention, control, and outcome) format. The aim of the following systematic review is to analyze TSHR expression level changes as a diagnosis and prognostic factor in papillary thyroid cancer.

Component Description Population PTC patients.

Intervention TSHR expression level and methylation status in PTC patients. Comparison Healthy patient group or other type of thyroid nodules.

Outcome TSHR expression level, as potential PTC early diagnostic and prognostic biomarker.

Search Strategy:

The search performed by using the following advanced MESH search on NCBI “MEDLINE” (PubMed), using google Chrome web browser. The key words and their combinations which used in the search:

11 ("thyroid"[All Fields] AND "cancer"[All Fields]) OR "thyroid cancer"[All Fields])) OR ("Thyroid cancer, papillary"[All Fields] OR "papillary thyroid carcinoma"[All Fields])) AND (("recurrence"[MeSH Terms] OR "recurrence"[All Fields]) OR ("prognosis"[MeSH Terms] OR "prognosis"[All Fields]) OR prognostic[All Fields] OR ("diagnosis"[All Fields] OR "diagnostic"[All Fields] OR "biomarker"[All Fields] OR " biomarker "[MeSH Terms] OR "marker"[All Fields]) OR marker [All Fields]) OR "expression"[All Fields] OR "methylation"[MeSH Terms].

To summarize, key words used in this search consisted of: (TSHR OR TSH receptor OR thyrotropin receptor) AND (papillary thyroid cancer OR papillary thyroid carcinoma) AND (recurrence OR prognosis OR prognostic OR diagnosis OR diagnostic OR expression OR methylation)”, thus using „AND” and „OR” as Boolean operators to narrow down the search. This search yielded 264 results with the final search performed without exclusion.

Screening (Initial inclusion and exclusion criteria)

Initial screening consisted of analyzing title and abstracts with specific inclusion and exclusion criteria that determine in advanced.

The inclusion criteria consisted of studies with at least: (i) Group of papillary thyroid carcinoma patients.

(ii) Thyroid carcinoma patients with a relation to TSH receptor expression. (iii) Papillary thyroid carcinoma patients with relation to diagnosis.

(iv) Papillary thyroid carcinoma patients with relation to prognosis.

(v) TSHR studies which performed quantitative expression measurements on PTC tissues. (vi) Qualitative or quantitative studies.

(vii) Articles published in English. (viii) Full-text articles.

(ix) Free articles.

(x) Publication date not older than 10 years.

Exclusion criteria consisted of:

(i) Non-PTC thyroid cancers studies.

(ii) In-vitro only studies without patient diagnostic or prognostic data. (iii)Single case-reports.

12 (v) Animal only studies.

(vi) Reviews, letters and abstracts presented in conferences.

(vii) Studies without TSHR prognostic or diagnostic statistical comparisons. (viii) Studies published before 2009.

After initial screening 205 articles were removed and remained 61 articles for eligibility for excluding articles by the abstract and full text. Two studies were found from additional sources and were included because they suited inclusion and exclusion criteria

Eligibility

13 Studies included in analysis (Supplementary Table 1)

According to the objectives, studies split into two groups for separate analyses. The two cohorts consisted of one group for analysis of the value of TSHR expression level changes (TSHR mRNA) in PTC patients and another group for analysis TSHR gene methylation status in PTC patients. Both groups will check the diagnostic and prognostic value of TSHR expression level changes in PTC but with different expression and aspects. Traditional circulating blood and tissue analysis and the relation of epigenetic status by new technology and methods.

Risk of Bias (Supplementary Table 2)

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SYSTEMIZATION AND ANALYSIS OF DATA

Fig. 1. Flow chart of the study selection Records identified through

PubMed database search (n = 264)

Additional records identified through other sources

(n = 2)

Records after duplicates removed (n = 266) Records screened (n = 266) Records excluded according inclusion/exclusion criteria1 (n = 205) Full-text articles assessed for eligibility

(n = 61)

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ANALYSIS OF DATA

PTC is the most common endocrine cancer and the number of incidence going up. In high risk patients FNA is the gold standard in the early diagnosis and prognosis follow up work of patient with PTC. In this systematic review, we analyse the significance of TSH receptor expression level and methylation status changes as diagnostic and prognostic biomarker in papillary thyroid cancer (PTC). This systematic review was done by grouping and qualitative assessing of all recent studies in this field.

Table 1: Evaluation of TSHR expression level changes as diagnostic biomarker for PTC patients by using quantitative mRNA detection methods in PTC tissue and peripheral blood. Author [Reference] TSHR expression analysis in tissue/ peripheral blood Discovery series Statistically significant correlation with diagnosis (P-value< 0.05) yes/no Conclusions Liu T, et al.[12]

tissue 172 PTC yes Expression levels of TSHR mRNA were significantly higher in well-differentiated thyroid cancer tissue especially in PTC comparing to healthy tissue. (93 patient with high expression of TSHR, 54.1%).

Liu TR, et al.[13]

tissue 150 PTC 21 HC

yes TSHR expression level was significantly higher in PTC patients comparing to healthy patients (102/150) 68% from PTC patients with higher TSHR expression level.

Tseng C-P et al.[17]

peripheral blood

129 PTC yes Relative high expression level of TSHR detected in peripheral blood for patient in early stage of PTC. So YK el al.[18] tissue 149 total (20PTMC, 20primary tumors

16 52metastat ic L.N, 57HC) Li YR et al.[19] peripheral blood

25 PTC yes Suggested that TSHR CEC (circulation endothelial cell) testing should be

performed at the initial diagnosis of PTC, due to evidence of high expression level of TSHR.

Aliyev et al.[21]

peripheral blood

152 PTmC yes Positive value of high TSHR expression level is associated with high prevalence of PTC (tall cell). Liu et al.[23] peripheral blood 104 PTC 11 HC

yes Suggested that TSHR mRNA high expression level can be used as a

biomarker for early diagnosis of PTC and PTmC. Lin et al.[24] peripheral blood 48 PTC 17 HC

yes TSHR CECs testing can be supplement method for disease monitoring and early diagnosis of PTC.

Kim et al.[26]

tissue N/A yes High expression of TSHR cause inhibition of defending molecular and physiological processes that can induce PTC.

Hirofumi el at.[29]

tissue 15 PTC 8 PDC 8 UDC

yes Observed that low expression level of TSHR correlates with PDC, authors conclude that increasing expression level of TSHR correlate with DTC specifically papillary thyroid cancer.

Gao et al.[31]

tissue 146 PTC yes Significant increased expression level of TSHR, promote and modulate PTC cancer cell proliferation.

Wang et al.[35]

tissue 27 PTC 56 HC

yes TSHR mRNA expression level was significantly lower in PTC compared to benign thyroid tissue.

Qiu et al.[40] peripheral blood 102 TC 34 PTmC 30 HC

yes High expression level of TSHR can be used as supplementary early diagnostic marker in preoperative PTmC patients which dramatically reduces unnecessary surgeries.

17 HC: healthy controls, N/A: not available.

18 Table 2: Evaluation of TSHR expression level changes and the potential prognostic value by using quantitative mRNA detection methods in PTC tissue and peripheral blood.

Author [Reference] TSHR expression analysed in tissue/ peripheral blood Potential prognostic value Statistically significant correlation with prognosis (P-value<0.05) yes/no Discovery series Conclusion Liu T, et al.[12] tissue distant metastasis and poor prognosis

yes 172 PTC Low TSHR expression level is associated with distant metastasis and poor prognosis.

Lung was the most common metastasis site.

Age>45 was important prognostic factor. Liu TR, et al.[13] tissue distant metastasis, recurrence yes 150 PTC 21 HC

High TSHR expression level is independent factor affecting the prognosis of PTC and might decrease developing of distant metastasis with specific target treatment (with specific target drug) in patient age>45 years. High expression of TSHR could inhibit metastasis by 45.3% and promote apoptosis of PTC cells after treatment.

Without treatment, high TSHR expression can predict poor prognosis and recurrence of PTC.

Su X, et al.[14] tissue Lymph node metastasis yes 300 PTC 252 HC

PTC patients with high expression level of TSHR variants were associated to develop lymph node metastasis. So YK el al.[18] tissue Lymph node metastasis yes 20PTmC, 20primary tumors,52 metastatic L.N,57HC

The presence of high TSHR expression level was observed to cause relative high frequency of sub clinical L.N metastasis in PTC patients (39/52)75%. Li YR et

al.[19]

peripheral blood

19 predicting recurrence in PTC patients.85.7% sensitivity. 77.8% specificity. Aliyev et al.[21] peripheral blood Lymph node metastasis yes 152 PTMC

Positive value of high expression level of TSHR mRNA is

associated with high prevalence of L.N metastasis involvement in patients age>45 (16% vs 10%). Liu et al.[23] peripheral blood recurrence yes 104 PTC 11 HC

Clinicians could use the high expressions level of TSHR mRNA predicting prognostic value as a biomarker for recurrence in PTC patient. Lin et al.[24] peripheral blood distant metastasis yes 48 PTC 17 HC

High expression of TSHR in CEC testing can be supplement method for disease monitoring and

prognosis status of PTC. Lin et al.[28] peripheral blood survival and remission

yes 128 Total High expression level of TSHR in CECs testing is useful prognostic tool for analysis overall survival and remission status by 76.6% in patient with DTC especially PTC.

Zhou et al.[37]

tissue Lymph node metastasis

yes 162 PTmC High TSHR expression level can be used as a supplementary marker predicting, invasion and lymph node metastasis in PTMC patients Aliyev et al.[38] peripheral blood recurrence yes 92 PTC 103 Total

Peripheral blood test of high TSHR mRNA level (>1.02) suggested to be a positive

biomarker in preoperative patients for predicting recurrence of differentiated thyroid cancer especially PTC. Sensitivity 70%. Specificity 76%. D’Agostino M, et al.[15] tissue Lymph node metastasis

yes 4 TC tumor aggressiveness is associated with low TSHR expression

PTC: papillary thyroid cancer, PTmC: papillary thyroid micro carcinoma, PDC: poorly differentiated cancer,

21 Correlation analysis between methylation status of TSHR gene and TSHR expression level changes in papillary thyroid cancer patients.

Table 3: TSHR gene promoter methylation status and the potential clinical value in PTC patients Author [Reference] Discovery series Methylation status of TSHR gene promoter in PTC patient (hyper/ methylation/ hypo) Potential clinical value Statistically significant correlation with clinical value (P-value<0.05) yes/no Conclusions Kartal K et al.[16] 69 Total 28 PTC Hypermethylation Diagnostic marker. Prognostic marker.

yes PTC patients group showed the highest TSHR

hypermethylation status rate comparing to controls 13/15 87%.

TSHR hypermethylation status could be used as early diagnosis biomarker in patient with well

differentiated thyroid cancer especially PTC, however it has limited value.

TSHR gene in the hypermethylated form is always present with PTC metastasis, therefore TSHR hypermethylation status may be used as a prognostic factor and potential disease

predictive factor in PTC. He H et

al.[20]

65 PTC Hypermethylation Diagnosis marker

yes TSHR gene hypermethylation cause gene silence and lower expression of TSHR.

This lead to predispose thyroid cell to dedifferentiate an essential step to towards malignant transformation.

Zheng et al.[22]

N/A Hypermethylation Prognostic marker

22 well as suppression of cell proliferation via mechanism of cell cycle that causes malignant process. Can help to determine therapeutic strategy for PTC. Landa et al.[25] 451 PTC 540 HC Hypermethylation Diagnostic marker

yes Identified potential diagnostic role of TSHR gene

methylation status within PTC patients. Mohammadi-Asl et al.[27] 25 PTC 25 HC Hypermethylation Diagnostic marker

yes TSHR gene promoter hypermethylation may be used as a biomarker in the pathogenesis and selecting patient with PTC, also related to TSHR low expression (TSHR gene down regulation). Khan et al.[30] 60 TC Hypermethylation Diagnostic marker

yes High implication of TSHR gene methylation status is associated with down

regulation of TSHR and PTC. Rosignolo et al.[32] 36 PTC Hypermethylation Diagnostic marker yes Downregulation of TSHR gene expression is common feature of TSHR gene methylation and PTC Smith et al.[33] 180 Total 32 PTC Hypermethylation Diagnostic Marker Prognostic marker yes TSHR promoter hypermethylation may be a biomarker for malignant thyroid cancer,

hypermethylation of TSHR promoter is more prevalent in PTC compared to other patients.

Patient with TSHR methylation occur tumor recurrence less frequently than patient with

unmethylated TSHR promoter region. Stephen et al.[34] 329 Total 85 PTC Hypermethylation Diagnostic marker

23 normal thyroid even in early stages of the disease.

Suggested as early diagnostic biomarker for detection of PTC. Also related to low expression of TSHR (down regulation).

24

DISCUSSION

26

CONCLUSION/RECOMMENDATIONS

In this study, we investigated the value of TSH receptor (TSHR) as early diagnostic and prognostic biomarker for PTC, TSHR expression level changes detected by using quantitative mRNA detection methods in PTC tissue and peripheral blood. Also evaluated the potential value of correlation between TSHR gene methylation status and TSHR expression level changes in PTC patients. TSHR expression level and TSHR gene methylation profile were reviewed and discussed for their diagnostic and prognostic value in papillary thyroid cancer patients. After review of all the studies, we can conclude that TSHR expression level changes have significant value as a independent diagnostic and prognostic biomarker in PTC patients. This marker may be promising novel biomarker for non-invasive, early detection and prediction of progression in PTC. TSHR expression level potentially can be used together with other clinicopathological features and traditional is used for early PTC diagnosis and prognosis.

Diagnostic significance of TSHR expression in PTC

PTC is the most common endocrine cancer and the number of incidence going up. In high risk patients FNA is the gold standard in the early diagnosis and prognosis follow up work of patient with PTC. Current guidelines suggest doctors to aspirate any nodule seems to be suspicious or taking sample during surgery. Traditional diagnostic methods usually associated with non-differential result, can be invasive, harmful in certain patients with a chance of complication. Studies showed positive association between high TSHR expression level and different stages and variants of PTC. It is suggested that TSH expression level changes is promising novel biomarker for non-invasive, early stages detection and diagnosis of PTC. Potentially it can be used alongside other diagnostic methods and prevent big number of unnecessary invasive methods and complications.

Prognostic value of TSHR expression with PTC patients

27 especially in patients older than 45 years. Moreover, TSHR gene hypermethylation causes downregulation of TSHR gene this can be used as a prognostic and potential predictive marker of L.N metastasis and recurrence of PTC.

TSHR gene methylation and the potential value in PTC.

Studies suggest positive value of TSHR gene promoter methylation status changes as a potential diagnostic and prognostic biomarker in PTC. Recent technological advances have allowed the identification of differently methylated regions. TSHR gene promoter methylation is frequently described as major repression mechanism regulating gene expression and play a central role in regulation of thyroid grow, thyroid functioning, tumor promoting effect and carcinogenesis. Although, the combination of gene methylation alterations, with other epigenetic changes may improve the clinical value. In conclusion, this systematic review found that methylation of TSHR gene promoter was associated with early diagnosis of PTC and predict poor prognosis with L.N metastasis, distant metastasis and PTC recurrence. Promoter methylation also demonstrated its ability to act as independent early diagnostic and prognostic biomarker for PTC.

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32

ANNEX:

Supplemental Table 1 – TSHR expression level studies included in the systematic review.

Author [Reference]

Year Region Method TSHR

expression Discovery series Liu T et al.[12] 2017 China qRT-PCR mRNA, 172 PTC Liu TR et al. [13] 2016 China RT-PCR mRNA 150 PTC 21 HC Su X et al.[14]

2019 China Genotyping promoter

methylation

300 PTC 252 HC D’Agostino

M et al.[15]

2014 Italy qRT-PCR, ChIP mRNA 4 PTC

Kartal K et al. [16]

2015 Turkey MSP, FNAC promoter

methylation 28 PTC 69 HC Tseng C-P et al.[17] 2017 Taiwan RT-PCR mRNA 129 PTC So YK el al.[18] 2012 Korea RT-PCR mRNA (149 TC) 20 single PTmC 20 PTC primary tumors 52 PTC metastatic L.N Li YR et al.[19] 2017 Taiwan RT-PCR mRNA 25 PTC He H et al.[20] 2014 USA qRT-PCR Gene expression 65 PTC Aliyev et al.[21] 2014 USA RT-PCR mRNA 152 PTmC Zheng et al.[22]

2015 China ChIP Promoter

methylation N/A Liu et al,[23] 2017 USA RT-PCR mRNA 104 PTC 11 HC Lin et al.[24] 2015 Taiwan RT-PCR mRNA 48 PTC 17 HC Landa et al.[25]

2013 Spain Genotyping Promoter

methylation 451 PTC 540 HC Kim et al.[26] 2014 South Korea RT-PCR mRNA N/A Mohamma di-Asl et al. [27]

2011 Iran COBRA Promoter

methylation

33 Lin et al.[28] 2017 Taiwan RT-PCR mRNA 128 HC Hirofumi el at.[29] 2008 Japan RT-PCR mRNA 15 PTC 8 PDC 8 UDC Khan et al.[30] 2014 India MSP Promoter methylation 60 TC Gao et al.[31] 2017 China RT-PCR mRNA 146 PTC Rosignolo et al.[32] 2015 Italy RT-PCR mRNA 36 PTC Smith et al.[33] 2008 London MSP Promoter methylation 180 HC 32 PTC Stephen et al.[34] 2017 USA MSP Promoter methylation 329 HC 85 PTC Wang et al.[35] 2011 China RT-PCR mRNA 27 PTC 56 HC Zheng et al.[36] 2017 China qRT-PCR mRNA 32 PTC 60 HC Zhou et al.[37] 2018 China RT-PCR mRNA 162 PTmC Aliyev et al.[38] 2015 USA RT-PCR mRNA 92 PTC 103 HC Gutnick et al.[39] 2012 USA RT-PCR mRNA 39 HC 8 PTC Qiu et al.[40] 2010 China RT-PCR mRNA 102 TC 34 PTmC 30 HC PTC: papillary thyroid cancer, PTmC: papillary thyroid micro carcinoma, HC: healthy controls,

qRT-PCR: Quantitative real-time reverse transcription polymerase chain reaction, ChIP: chromatin immunoprecipitation, COBRA: combined bisulfite restriction analysis,

34 Supplemental Table 2.1 – Risk of bias of individual studies

Author S u X Zh o u L iu T T se n g C -P L i Y R Li u Li n G ao step h en L iu T R K ar tal K Zh en g Li n Ro si gn o lo Al iy ev Year 2019 2018 2017 2017 2017 2017 2017 2017 2017 2016 2015 2015 2015 2015 2015 Criteria Selection Bias Adequate Case Definition * * * * * * * * * * * * * * * Representativeness of the cases * * * * * * * * * * * * * * * Selections of controls * * * * * * * * * * * * * * Definition of Controls * * * * * * * * * * * * * * Comparability * * * * * * * ** ** * * * ** * * Exposure Ascertainment of exposure * * * * * * * * * * * * * * * Same method of ascertainment for cases

and controls

* * * * * * * * * * * * * * *

Non-response rate * * * * * * * * * * * * * * *

35 Cont. Supplemental Table 2.2 –Risk of bias of individual studies

Author D A gost in o M H e H Al iy ev K im kh an La n d a S o Y K G u tn ic k M o h am m ad i As l W an g Q iu Hi ro fu m i Sm ith Year 2014 2014 2014 2014 2014 2013 2012 2012 2011 2011 2010 2008 2008 Criteria Selection Bias

Adequate Case Definition * * * * * * * * * * * * * Representativeness of the cases * * * * * * * * * * * * * Selections of controls * * * * * * * * * * * Definition of Controls * * * * * * * * * * * Comparability * * * * * * ** * * * * ** * Exposure Ascertainment of exposure * * * * * * * * * * * * * Same method of ascertainment for cases

and controls

* * * * * * * * * * * * *

Non-response rate * * * * * * * * * * * * *