Accepted Manuscript

V600E
Imbalance in DNA repair machinery is associated with BRAF mutation and
tumor aggressiveness in papillary thyroid carcinoma

Bruna S. Lutz, Natalia M. Leguisamo, Nicole K. Cabral, Helena C. Gloria, Keli C.

Reiter, Grasiela Agnes, Virgilio Zanella, Erika L.S. Meyer, Jenifer Saffi
PII: S0303-7207(17)30632-9
DOI: 10.1016/j.mce.2017.12.004
Reference: MCE 10143

To appear in: Molecular and Cellular Endocrinology

Received Date: 17 July 2017

Revised Date: 19 November 2017
Accepted Date: 7 December 2017

Please cite this article as: Lutz, B.S., Leguisamo, N.M., Cabral, N.K., Gloria, H.C., Reiter, K.C.,
Agnes, G., Zanella, V., Meyer, E.L.S., Saffi, J., Imbalance in DNA repair machinery is associated with
V600E
BRAF mutation and tumor aggressiveness in papillary thyroid carcinoma, Molecular and Cellular
Endocrinology (2018), doi: 10.1016/j.mce.2017.12.004.

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 ACCEPTED MANUSCRIPT
Imbalance in DNA repair machinery is associated with BRAFV600E mutation

and tumor aggressiveness in papillary thyroid carcinoma

Bruna S. Lutza, Natalia M. Leguisamo a,b

, Nicole K. Cabrala, Helena C. Gloriaa,

Keli C. Reiterc; Grasiela Agnesd; Virgilio Zanellae, Erika L. S. Meyere#, Jenifer

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Saffia*#

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a
Laboratory of Genetic Toxicology, Universidade Federal de Ciências da Saúde

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de Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil.
b
Laboratory of Molecular and Cellular Cardiology, Instituto de

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Cardiologia/Fundação Universitária de Cardiologia (IC/FUC), Porto Alegre, Rio
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Grande do Sul, Brazil.
c
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Laboratory of Pathology, Universidade Federal de Ciências da Saúde de Porto

Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil.

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d
Laboratory of Molecular Biology, Universidade Federal de Ciências da Saúde
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de Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil.

e
Thyroid Section, Endocrine Division, Santa Casa de Misericórdia de Porto
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Alegre (ISCMPA), Porto Alegre, Rio Grande do Sul, Brazil.

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*To whom correspondence should be addressed: Laboratory of Genetic

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Toxicology, Universidade Federal de Ciências da Saúde de Porto Alegre

(UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil. E-mail:

jenifers@ufcspa.edu.br. Tel: +55 51 33038861.

#
These authors shared senior authorship.
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Abstract

The involvement of alterations in MLH1, an essential mismatch repair

component, in BRAFV600E mutated papillary thyroid carcinoma (PTC) has been

suggested to be associated with features of tumor aggressiveness. Thirty-two

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PTC and surrounding normal thyroid tissues were evaluated for 11

representative DNA repair genes expression. BRAFV600E mutational status

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assessment and clinicopathological correlations were evaluated for their gene

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and protein expression. BRAFV600E PTC is associated with lower levels of XPD

and MLH1 gene expression. Decrease in MLH1 and XPD mRNA levels in

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BRAFV600E PTC (but not their protein products) are associated with predictors of
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poor patient outcomes. Considering the complete subset of patients, MGMT
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and XRCC2 genes were shown down and upregulated, respectively, in PTC

tissues. Low expression of MGMT gene and weak XRCC2 protein expression
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were correlated with characteristics of tumor aggressiveness. These results

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suggest that an imbalance in DNA repair gene expression in PTC is associated

with aggressive clinicopathological features and BRAFV600E mutation.

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Keywords: papillary thyroid cancer; BRAFV600E; DNA repair; prognosis.

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Highlights

• BRAFV600E mutation is the main genetic alteration related to PTC.

• The role of DNA repair mechanisms in thyroid cancer remains largely

unknown.

• Low mRNA levels of MLH1 and XPD, but not their protein products, are

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associated with BRAFV600E mutational status and aggressive

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clinicopathological features.

• MGMT and XRCC2 are differentially expressed in PTC

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• Low expression of MGMT gene and weak XRCC2 protein expression

were correlated with characteristics of tumor aggressiveness.

•
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Transcriptional level of DNA repair components may be part of the
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malignant process in BRAF mutation-promoted thyroid tumorigenesis of
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PTC.
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1. Introduction

Papillary thyroid carcinoma (PTC) is responsible for up to 95% of

malignant thyroid tumors and the majority of patients have a favorable outcome

(Zhu et al., 2015; Howlader et al., 2016). However, 15% will develop local tumor

recurrence and 5 to 10% will presentdistant metastasis (Mazzaferri and Jhiang,

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1994; Sherman, 2003). Multiple genetic and epigenetic alterations have been

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described in thyroid cancer in recent decades. Mutations in RET/PTC, RAS, or

BRAF, which result in constitutive mitogen-activated protein kinase (MAPK)

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signaling, are found in approximately 70% of PTC tumors with little overlap

between mutated genes (Kimura et al., 2003). More recently, TERT has been

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suggested as a new oncogene in thyroid cancer and its association with
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BRAFV600E mutation has a robust synergistic impact on the aggressiveness of
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PTC (Xing et al., 2014; Liu and Xing, 2016).

BRAFV600E is the most common genetic alteration found in PTC, with a

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worldwide prevalence ranging from29 to 83% (Davies et al., 2002; Ciampi and
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Nikiforov, 2007; Oler and Cerutti, 2009). In colon cancer, BRAF mutation have

been associated with genetic instability and aberrant hypermethylation of some

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DNA repair genes, such as the mismatch repair gene Human Mut-L Homologue

1 (MLH1) (de Vogel et al., 2009). Previous studies that examined the
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methylation status of a large number of DNA repair genes in human thyroid

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cancer cell lines and PTC tissues showed that aberrant methylation of the

MLH1 gene was associated with lymph node metastasis and BRAF mutation in

PTC (Guan, 2008). In addition, recently studies revealed that thyroid tumors

with point mutations (BRAF, IDH1 and RAS) show a decrease in MLH1

expression (Santos et al., 2013).

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DNA repair genes protect genetic integrity in normal cells by mismatch

repair, nucleotide excision, base excision, and double-strand break repair (Lord

and Ashworth, 2012). Impaired DNA repair is related to increases in mutation

frequency, genomic instability and cell death. Moreover, polymorphisms in DNA

repair genes have been associated with development of thyroid cancer

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(Gatzidou et al., 2010; Chiang et al., 2008; Hu et al., 2013; Wu et al., 2014;

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Shkarupa, et al., 2015; Sturgis et al., 2005; Yan et al., 2016).

Fewimmunohistochemical no-paired studies have evaluated DNA repair

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expression profile in benign and malignant thyroid

neoplasiasdemonstratingchanges inexpression protein is associated with

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clinicopathological characteristics of greater aggressiveness, such as larger
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turmor size and presence of lymphatic and vascular invasion (Ruschenburg et
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al., 2006; Giaginis et al., 2011). These findings suggest a role of DNA repair

genes in thyroid tumorigenesis and a possible association betweenthese

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pathways with BRAF mutation.

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Considering the lack of studies focusing more widely on DNA repair

genes expression in PTC samples, in the current study, we examined the profile
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of DNA repair gene expression in PTC tissues and analyzed its correlation

withBRAFV600E mutation and clinicopathologiccharacterisctics of PTC.

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2. Materials and methods

2.1. Patients and tissues

Tumor and surrounding normal thyroid tissue were collected

consecutively from 32 patients diagnosed with PTC attending the Endocrine or

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Head and Neck Surgery Divisions at Irmandade da Santa Casa de Misericórdia

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de Porto Alegre – Brazil. Tissue samples were collected in tumors above 1 cm

of diameter. Surgery was independently indicated by attending physicians

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based on clinical indication. Informed consent from all participants and

clearance from the Ethical Committees of the participating Institutions were

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obtained (approval numbers 331.061 and 362.887). We excluded patients who
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had a history of head and neck irradiation or received chemotherapy prior to
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surgery. Specimens were frozen in liquid nitrogen after surgical resection and

stored at 80°C. Experienced pathologists confirmed final histological

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classification from paraffin-embedded sections. Tumors were histological

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classified according to WHO recommendations (DeLellis et al., 2004). The

clinical stage was determined by the Tumor/Node/Metastasis (TMN) system

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(Egner et al., 2010). The risk of recurrence was classified according to the

American Thyroid Association (ATA) (Cooper et al., 2009). The information

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obtained from this study did not influence or affect the patients’ diagnosis or
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treatment.

2.2. Extraction of nucleic acids and reverse transcription

Total RNA was extracted from 30 mg of frozen tumors and surrounding

normal thyroid tissues by RNeasy® mini kit (QIAGEN GmbH, Hilden, Germany)
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including a DNAse treatment with RNase-Free DNase Set (QIAGEN GmbH,

Hilden, Germany). To enrich the tumor specimens, regions of high cellularity

were obtained from the central region of the tumor. For DNA extraction the

tissue paraffin-embedded blocks were sectioned at 5 µm without any stain or

fixer and the thyroid cancer cells were scraped from the glass slides using a

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surgical blade. Then the DNA was obtained according the manufacturer’s

protocol of MagneSil® Genomic, Fixed Tissues System (Promega Corporation,

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Madinson, WI, USA). The amount of the isolated nucleic acids was determined

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through spectrophotometry by BioSpec-Nano (Shimadzu Corporation, Kyoto,

Japan) and the integrity of nucleic acids was checked using gel electrophoresis.

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The reverse transcription was performed from 1 µg of RNA by RT² PCR Array
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First Strand Kit (QIAGEN Sciences, Maryland, USA) according to the
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manufacturer’s recommendations.
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2.3. BRAFV600E mutation analysis

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The exon 15 of the BRAF gene was amplified by polymerase chain

reaction (PCR) through Platinum®Taq DNA Polymerase Kit (Invitrogen by Life

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technologies, São Paulo, Brazil) and appropriate primer pairs: forward 5′-

CTTCATAATGCTTGCTCTGATAGGA-3′ and reverse 5′-

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CAGGGCCAAAAATTTAATCAGTGGA-3′. PCR products were purified with the

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Ilustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,

Buckinghamshire, UK) as specified by the manufacturer. Sanger sequencing

reaction was performed with the BigDye Terminator V3.1 Cycle Sequencing Kit

(Life Technologies Corporation, Carlsbad, CA, EUA) on an ABI PRISM 3130

genetic analyzer (Applied Biosystems, Foster City, CA, EUA). The presence of
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BRAFV600E mutation was confirmed in both forward and reverse directions by an

independent PCR amplification and sequencing reaction.

2.4. Quantitative real-time PCR

Differential DNA repair gene expression between tumor and surrounding

normal thyroid tissues were assessed by quantitative real-time reverse

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transcription-PCR (RT-PCR) on an Applied BiosystemsStepOnePlus™ Real-

Time PCR System (Applied Biosystems, Foster City, CA, USA) using the RT2

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RNA QC PCR Array (PAHS-999Z C-1, QIAGEN Sciences, Maryland, EUA)

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quality control plates and the custom RT2 Profiler™ PCR Array (CAPH-13265,

QIAGEN Sciences, Maryland, EUA).The pathways and the representative

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genes selected to compose this predesigned assay are detailed as follows:
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direct repair (MGMT, NM_002412), mismatch repair (MMR) (MLH1,
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NM_000249; MSH2, NM_000251), base excision repair (BER) (APE1,

NM_080649; OGG1, NM_002542; XRCC1, NM_006297), nucleotide excision

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repair (NER) (XPD, NM_000400), homologous recombination pathway (HR)

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(RAD51, NM_002875; XRCC2, NM_005431; XRCC3, NM_005432) and non-

homologous end joining recombination (NHEJ) (KU80, NM_021141). Data

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analysis was based on the ∆∆Ct method and the Ct values were normalized

according to the expression of ACTB and B2M in each sample.

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2.5. Immunohistochemistry studies

For protein expression analysis one representative paraffin-embedded

block from each case containing tumor tissue was selected. The blocks were

sectioned at 4µm and submitted to a routine immunohistochemical technique,

which included deparaffinization and rehydration, antigen retrieval, inactivation

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of endogenous peroxidase and blockage of non-specific reactions. The primary

antibodies incubated overnight at 4°C were monoclonal mouse anti-MGMT

(1:100 dilution; #ab81705; Abcam, Cambridge, MA, USA), polyclonal rabbit anti-

XRCC2 (1:100 dilution; #PA5-32643; Thermo Fisher Scientific, USA),

monoclonal rabbit anti-MLH1 (1:100 diluition; #ab92312; Abcam, Cambridge,

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MA, USA) and monoclonal rabbit anti-XPD (1:100 diluition; #ab167418; Abcam,

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Cambridge, MA, USA). To follow the assay, slides were incubated with the

secondary and tertiary antibodies for 30 min each at room temperature (Reveal

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Polyvalent HRP-DAB Detection System, Spring Bioscience, CA, EUA). Normal

human tonsil tissue and human testis cancer tissue, respectively, were used as

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a positive control. The negative control was obtained by omission of the primary
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antibody. The reaction products were visualized by DAB. Slides were
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counterstained with hematoxylin.

Protein expression was evaluated using immunohistochemistry images of

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all PTC tissue samples, captured using a DM6 Leica coupled to LAS X system
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of image capture software (Leica Microsystems GmbH, Wetzlar, Germany) at

200× magnification. Five hot spots fields were randomly selected and
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analyzedthrough ImageJ Version 1.50i (National Institutes of Health, Bethesda,

Maryland) by two blinded observers. All background area was removed by

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moving brightness and hue sliders. To measure only stained areas, the
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brightness slider was increased without changing the hue slider, and the

immunohistochemistry stained areas were selected. Mean and stained area

were recorded to the excel sheet (Sati, Soygur and Celik-Ozenci,2016). The

percentages of positively stained follicular cells were obtained by the

percentage of the stained area with a given intensity. Specimens were

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considered positive when more than 5% of the follicular cells were stained. The

immunoreactivity of the follicular cells for MGMT and XRCC2 was scored

according to the percentage of stained area as 0: negative staining - 0–4% of

tumor positivity; 1: 5–24% of tumor positivity; 2: 25–49% of tumor positivity; 3:

50–100% of tumor positivity. Its intensity was classified as it follows:0, negative

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staining; 1, mild staining; 2, intermediate staining; 3, intense staining, based on

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intensity mean. Finally, both values were multiplied together, and the staining

score was stratified as weak (score range, 0–2) or strong (score range ≥3)

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according to the proportion and intensity of positively stained tumor cells. Both

nuclear and cytoplasmic immunostaining were considered to analysis (Giaginis

et al, 2011).
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2.6. Statistical analysis

Statistical analysis was performed by SPSS 17.0 (SPSS Inc., Chicago,

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IL, USA) statistical software. Descriptive statistics include frequencies and

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corresponding percentages for binary variables and means for continuous

variables. The difference in RNA expression between paired tumor and normal
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specimens was evaluated using Wilcoxon matched-pair test. To analyze the

association between protein expression and clinicopathological characteristics

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the chi-square test was applied, followed by Exact Fisher test. The correlation of
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fold change and clinicopathological characteristics was evaluated with either the

Mann-Whitney or the Kruskal-Wallis tests. Chi-square test was also conduced

to assess the associations between BRAFV600E mutation and clinicopathological

characteristics. In all analyses, a two-tailed P<0.05 was considered statistically

significant.
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3. Results

3.1. Patients and BRAFV600E analysis

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Clinical and pathological characteristics of the 32 patients included in this

study according to BRAFV600E mutation are detailed in Table 1. The mean age at

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the diagnosis was 39.9 +/- 14 years. The median size of tumor was 2.0 cm. All

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patients presented normal serum TSH levels (0.4 – 4.0 UI/L) at surgery. The

BRAFV600E mutation was found in 19 of 32 cases (59.0%). The mutational status

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was not associated with clinicopathological characteristics (Table 1).
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3.2. Low mRNA levels of MLH1 and XPD genes were correlated with BRAFV600E
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mutation and clinicopathological characteristics of tumor prognosis

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The profile of DNA repair genes expression according to BRAFV600E

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mutational status is shown in Figure 1. Of all DNA repair genes studied, we

found a significant association between BRAFV600E mutation and low expression

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of MLH1 (p=0.037) and XPD (p=0.025) genes (Figure 2). Interestingly,

reduction in mRNA levels of MLH1 and XPD in tumor samples in relation to

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surrounding normal tissues were associated with presence of lymph nodes

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metastasis (p=0.014 and p=0.014) and increased risk of recurrence (p= 0.006

and p=0.006), respectively. In addition, lower tumor mRNA levels of MLH1 were

associated with TNM stage III (p=0.003) and minimum local invasion (p=0.022).

In another way, tumors expressing reduction of MLH1 gene expression were

small than 2 cm in diameter (p=0.027) (Table 2). A significant reduced

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expression of XPD was observed in classical variant of PTC in comparison to

normal surrounding tissue (p=0.029) (Table 2).

The analysis of protein expression in PTC demonstrated a strong MLH1

immunoreactivity in 19 (59%) out of 32 samples (p=0.075). A weak XPD

expression was observed in 24 (75%) out of 32 PTC samples (p<0.01). Strong

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protein expression of MLH1 was related to classical variant of PTC (p=0.036).

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We did not find significant correlations between MLH1 and XPD protein

expression and others clinicopathological features except for a trend of

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correlation between MLH1 and XPD expression and presence of lymph node

metastasis (p=0.061 and p=0.059, respectively) (Table 3). Figure 3

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demonstrates representative images of strong and weak expression of MLH1
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and XPD, respectively in PTC tissue compared to their expression in
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surrounding normal tissue.

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3.3. MGMT and XRCC2 genes are differentially expressed in PTC samples
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Among the 11 representative DNA repair genes selected analyzed in

PTC samples, only 2 presented differential gene expression between PTC and
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respective surrounding normal tissue (Figure 4A). The direct repair gene,

MGMT, showed a lower expression level in thyroid tumor tissues in comparison

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to matched normal tissues (p=0.038), with a 0.36-fold decrease (p<0.05). On

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the other hand, regarding the homologous recombination pathway, XRCC2,

presented its mRNA levels significantly higher in thyroid tumor tissues than in

the normal tissues (p=0.003) with a 0.53-fold increase (p<0.05). Relative mRNA

expression of the pathway genes with differential expression is shown in Figure

4B.
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3.4. Low mRNA levels of MGMT are associated with clinicopathological

characteristics of PTC

Focusing on DNA repair genes with differential expression, we observed

a low MGMT fold change was significantly correlated with tumor size less than 2

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cm (p=0.040), presence of lymph node metastasis (p=0.003) and intermediate

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risk of recurrence (p=0.022) (Figure 5). On the other hand, no correlation was

found to XRCC2 higher mRNA levels on tumor tissue and clinicopathological

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characteristics. All associations between others representative genes of six

distinct DNA repair pathways (namely, direct repair, MMR, BER, NER, HR and

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NHEJ) and clinicopathological characteristics are described in Supplementary
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Table 1.
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The analysis of protein expression in PTC tissues demonstrated a strong

MGMT immunoreactivity in 23 (72%) out of 32 samples. A weak XRCC2

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expression was observed in 22 (69%) out of 32 samples. We did not find

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significant correlations between MGMT and XRCC2 protein expression and

clinicopathological features, except for a correlation between XRCC2

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expression and minimum local invasion (p=0.018) (Table 4). Figure 6

demonstrates representative images of strong and weak expression of MGMT

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and XRCC2, respectively in PTC tissue compared to their expression in

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surrounding normal tissue.

4. Discussion

In the present study, we analyzed the profile of DNA repair genes in

samples of PTC according to BRAFV600E mutation. We demonstrated that

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reduction in mRNA levels of MLH1 and XPD, but not the changes in their

protein products, are associated with BRAFV600E mutation, presence of lymph

node metastasis and intermediate recurrence risk, which are indicative of tumor

aggressiveness. Our study also demonstrated that MGMT mRNA levels are

significantly reduced in PTC samples and it is correlated with presence of

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metastatic lymph nodes and intermediate risk of recurrence, but the respective

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protein content does not correlate with these clinicopathological features.

According to our knowledge, we are reporting for the first time that XRCC2, a

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representative component of homologous recombination pathway, is

upregulated in PTC tissues. Despite this alteration was not followed by

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increases in protein content, PTC samples with weak XRCC2 immunoreactivity
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presented minimum local invasion.

BRAFV600E mutation was present in a significant proportion (59%) of our

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sample, but without correlation with clinicopathological characteristics, which

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can be attributed to the limited number of tumor samples included in this study.
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In fact, the association of BRAFV600E with the clinical manifestations and

prognosis of PTC remains controversial (Cappola and Mandel, 2013; Xing et al.,
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2013). As previously reported, we also found a diminished expression of MLH1

in patients harbouring BRAFV600E mutation (Santos et al., 2013). Interestingly,

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considering our results, we strengthen the association of DNA repair machinery

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and BRAF mutation in PTC.

Thyroid epithelial cells are constantly exposed to H2O2 generated during

thyroid hormone biosynthesis, which increases the disponibility of –OH free

radicals and create the conditions for DNA oxidation and subsequent lesions,

such as single and double strand breaks (Driessens et al., 2009). The oxidative
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burden measureable in the normal thyroid gland is probably associated to

hormone synthesis and H2O2 production (Krohn et al., 2007). Recently, it was

reported that MLH1 was downregulated in PTC in comparison to normal

paratumoral tissue, but upregulated when in comparison to normal thyroid

(adjacent to benign nodules) (Javid et al. 2017). This data points out to a

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possible role of defective DNA repair in the context of excessive oxidative stress

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in the malignant process of PTC tumorigenesis.

However, the mechanism that induces the reduction of MLH1 gene

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expression is not clear. Investigations of MLH1 gene methylation status in PTC

did not provided conclusive findings so far. Aberrant DNA methylation and

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expression silencing is an important molecular alteration detected in DNA repair
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genes in different types of cancer. Guan et al. have demonstrated a frequent
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hypermethylation of the mismatch repair gene MLH1 and its close association

with lymph node metastasis and, as noted in colon cancer, BRAF mutation in
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PTC (Guan et al., 2008). In counterpart, another study did not find significant
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differences in MLH1 promoter methylation between benign and malignant

thyroid lesions, suggesting that mechanisms other than DNA methylation in the
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CpG islands could be acting to silence the expression of MLH1 in PTC (Santos

et al., 2013).
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Focusing on the clinical relevance of MLH1 expression, it is interesting to

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note that even in a small number of samples studied, a decrese in MLH1 mRNA

levels demonstrated an important association with lymph nodes metastasis,

minimum local invasion, TNM stage III and intermediate recorrence risk. In

counterpart, lower expression of MLH1 was associated with small tumors

(<2cm), indicating that the tumor size may not be a perfect clinical predictor of
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prognosis. Awkwardly, linearity between mRNA levels and protein expression

was not achieved for MLH1 (spearman’s rho = 0.394; p=0.095. Data not

shown.), which may denote that changes in MLH1 transcripts’ levels are

possibly related to the cellular transitioning to a more aggressive profile, but the

final protein product is not essential for the maintenance of this condition.

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However, moderate/strong MLH1 immunoreactivity is more frequently

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observed in malignant compared to benign thyroid lesions (Giaginis et al.,

2011). It was also reported that moderate/strong MLH1 immunoreactivity is

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associated with the presence of lymphatic and vascular invasion (Giaginis et al.,

2011). Despite we did not find correlations between MLH1 protein expression

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and the clinicopathological features, except for a trend with presence of lymph
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node metastasis (p=0.061), these data suggest that MLH1 could be a strong

and independent of BRAFV600E mutational status molecular marker of tumor

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aggressiveness in PTC. Nevertheless, the real prognostic value of such findings

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needs to be analyzed in follow-up studies involving larger number of patients.

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For the first time, we reported BRAFV600E association with XPD (NER

pathway) down-regulation in PTC. Similar to MLH1 gene expression clinical

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associations, lower mRNA levels of XPD were associated with presence of

lymph nodes metastasis and increased recurrence risk. However, despite XPD
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protein expression was found to be weak in nearly 80% of the cases, it did not
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associate with clinicopathological features, except for a trend of association with

lymph node metastasis (p=0.059). Recently, low XPC was correlated with BRAF

and NRAS mutation in melanoma samples (Davey, 2016). It has been

suggested that NER and MMR pathways influence the DSB repair by three

steps: physical facilitation of HR or NHEJ machinery through the processing of

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intermediates during DSB repair; participation in primary damage surveillance;

and signaling between primary damage reorganization, induction of cell cycle

checkpoint and permission for cell cycle progress (Zhang, Rohde and Wu,

2009). We suggest that the correlation between BRAFV600E and MLH1 or XPD

reduced expression may be a step of the malignant process involving XRCC2

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increased activity. Another noteworthy result of our study is regarding the

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increased expression of XRCC2 mRNA levels in PTC comparing with

surrounding normal thyroid tissue. XRCC2 is a HR pathway gene that encodes

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a member of the Rad51-like protein (Thacker et al., 1995; Johnson, Liu and

Jasin, 1999). XRCC2 contains the ATP binding domain known as Walker motifs

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A and B and its cell-deficiency results in a decrease of up to 100-fold of HR
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repair system capacity (Walker et al., 1982; Johnson, Liu and Jasin, 1999).
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XRCC2 mRNA levels were not associated with tumoral features, but a

correlation of weak XRCC2 protein expression with minimum invasion was

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identified. In primary colorectal tumors, the levels of XRCC2 were significantly

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higher than that in the matched adjacent normal tissues. It was associated with

tumor site, Dukes’ and Tumor-nodes-metastasis (TNM) staging. The authors

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also demonstrated that XRCC2 upregulation inhibited CRC cell apoptosis and

promoted proliferation enriching cells in the G0/G1 phase (Xu et al., 2014). It is
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possible due to a non-conservative substitution or deletion of amino acid 188,

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which can affect cellular sensitivity to DNA damage (Liu et al., 1998; Rafii et al.,

2002).

Underexpression and hypermethylation of MGMT has been associated

with tumorigenesis in several human cancer tissues, such as gastric carcinoma,

thymic epithelial tumor, ductal breast carcinoma and colorectal cancer, leading
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to cell malignancy, modulation of chemotherapy responses and numerous

clinical outcomes (Bardhanand Liu, 2013; Mokhtar et al., 2014; Yousuf et al.,

2014; Asiaf et al., 2015, Leguisamo et al., 2017). Previous unpaired

immunohistochemical studies have demostrated that negative/weak MGMT

immunoreactivity was significantly more frequently observed in malignant

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compared to benign thyroid lesions. However, the majority of PTC samples

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(67%) showed moderate to strong immunoreactivity for MGMT, a similar finding

observed in our study (72% of PTC samples) (Giaginis et al., 2011). In

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consonance with Giaginis et al., we did not find correlation between MGMT

protein expression and clinicopathological parameters. In another way, we

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found that the mRNA levels of MGMT are significantly reduced in PTC
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comparing with surrounding normal thyroid tissues and the decrease of mRNA
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levels were correlated with tumor size, lymph node metastasis and recurrence

risk Unexpectedly, MGMT gene and protein expression in PTC samples were
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not linear (spearman’s rho = - 0.297; p= 0.191. Data not shown). The
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mechanism that regulates MGMT protein translation is still not clear. It has been

considered that MGMT protein levels may be reliant on an alternative

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translation mechanism. This was demonstrated during conditions of reduced

global translation rates, when also occurs a decrease of mRNA association with
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polysomes. Thus, MGMT mRNA remains associated with heavy

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polysomes. This is strong evidence that MGMT mRNA might be translated

using a cap-independent mechanism, which implies in lower levels of mRNA,

but constant translation. (Gandin et al., 2014; Smalley et al, 2014)

In fact, loss of MGMT is defined as “field defect”, which means it is not

the only responsible but contributes to the progression of cancer (Nagasaka et

 ACCEPTED MANUSCRIPT
al., 2008), which can explain the correlation between MGMT lower levels and

smaller tumors, indicating that loss of this gene may be an initial event in thyroid

tumorigenesis. Thus, the reduced expression of MGMT in PTC is in accordance

with other human tumors and it corroborates the assumption that direct repair

can play a role in both development and progression of thyroid cancer.

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Our results characterize the expression of DNA repair genes in PTC

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tissues, with alteration in key-components of DR (MGMT) and HR (XRCC2)

pathways. The association of BRAFV600E with MMR (MLH1) and NER (XPD)

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pathway genes suggests that the transcriptional alterations in DNA repair genes

may be part of the malignant process particularly in BRAF mutation-promoted

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thyroid tumorigenesis of PTC. However, since the protein contents of these
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genes did not confirm association with clinico-pathological characteristics of
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poor prognosis, it is possible that these transcriptional alterations may be part of

the malignant process in BRAF mutation-promoted thyroid tumorigenesis of

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PTC.
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Acknowledgements

We thank Suzana Elisabete Lamonatto, Renata Fragomeni Almeida and

Karen Margarita Rico Escamilla for assistance in collecting tissues samples.

Funding

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This work was supported by the Brazilian Funding Agencies “Fundação

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de Apoio à Pesquisa do Rio Grande do Sul” – PPSUS –

FAPERGS/MS/CNPq/SESRS [grantnumber 002/2013] and “Conselho Nacional

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de Desenvolvimento Científico e Tecnológico” – MCTI/CNPQ/Universal

[14/2014].

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Disclosure statement
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The authors declare that there is no conflict of interest that could be

perceived as prejudicing the impartiality of the research reported.

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Table titles

Table 1: Clinicopathological characteristics of patients according BRAFV600E

Table 2: Correlation between MLH1 and XPD gene expression and

clinicopathological characteristics of PTC.

Table 3: Frequency of MLH1 and XPD proteins levels and its correlation with PTC

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clinicopathological features

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Table 4: Frequency of MGMT and XRCC2 proteins levels and its correlation

with PTC clinicopathological features.

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Figure legends AN
Figure 1: Heat map of individual DNA repair gene expression changes and
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BRAFV600E mutational status in papillary thyroid carcinoma samples. Fold

changes were calculated for neoplastic tissue vs. adjacent normal tissue. Gray
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indicates the presence and white indicates the absence of the mutation. Blue
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indicates decreased relative gene expression and red indicates increased

relative gene expression.

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Figure 2: Low MLH1 and XPD expression between tumor and surrounding
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normal thyroid tissues is associated with BRAFV600E mutation. The

statistical significance was determined by Mann-Whitney test.

Figure 3: Representative photomicrographs showing immunohistochemical

staining in papillary thyroid carcinoma (T) and adjacent normal tissue (N) for

MLH1 and XPD. Images were taken at x400 magnification.

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Figure 4: Gene expression analysis of DNA repair genes in matched tumor

and normal tissues. (A) Relative fold change (tumor/normal tissue) in the

mRNA expression level was shown as the logarithmic scale of relative fold

change in expression (RQ) 2-∆∆CT values. The statistical significance of the

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observed fold change in expression was determined by Mann-Whitney test.

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*represent statistically significant difference (p<0.05). (B) mRNA levels (∆CT) of

MGMT and XRCC2 genes. The statistical significance of the observed change

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in mRNA expression was determined by Wilcoxon matched-pair test.

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Figure 5: Low MGMT expression between tumors and surrounding normal
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thyroid tissues is significantly associated with smaller tumors, lymph
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node metastasis and intermediate risk of recurrence. Horizontal lines

represent the median; the bottom and top of the boxes represent the 25th and
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75th percentiles, respectively; and the vertical bars represent the range of data.
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The statistical significance was determined by Mann Whitney test.

Figure 6: Representative photomicrographs showing immunohistochemical

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staining in papillary thyroid carcinoma (T) and adjacent normal tissue (N) for

MGMT and XRCC2. Images were taken at x400 magnification.

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Supplementary Material information

Supplementary Table 1: Changes in the expression of representative DNA repair

pathway genes are associated with clinicopathological characteristics in PTC patients.

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Table 1. Clinicopathological characteristics of patients
according BRAFV600E.
Clinicopathological Wild-type P
BRAF V600E
features BRAF value
Total cases 19 (59%) 13 (41%)
Age (years) 41.95±15.20 37.00±11.85 0.617
Tumor size (cm) 2.34±1.22 2.28±1.61 0.336
Sex

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Female 15 (47%) 13 (41%)
0.077
Male 4 (13%) 0 (0%)
Histological variants

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of PTC
Classic 14 (44%) 4 (12.5%)
Follicular 4 (12.5%) 7 (22%)

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0.091
Microcarcinoma 1 (3%) 1 (3%)
Warthin-like 0 (0%) 1 (3%)
Multifocality

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Present 6 (19%) 2 (6%)
0.299
Absent 13 (41%) 11 (34%)
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Minimum local
invasion
Present 15 (47%) 22 (7%)
0.132
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Absent 4 (12.5%) 6 (19%)

Lymph node
metastasis
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Present 6 (19%) 2 (6%)

0.299
Absent 13 (41%) 11 (34%)
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AJCC/TNM stage
I-II 14 (44%) 12 (37%)
0.108
III 5 (16%) 1 (3%)
Risk of recurrence
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Low 3 (9%) 6 (19%)

0.061
Intermediate 16 (50%) 7 (22%)
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Table 2. Correlation between MLH1 and XPD gene expression and clinicopathological characteristics
of PTC.
MLH1 XPD

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High Low P High Low P
expression expression value expression expression value

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Total (n, %) 16 (50) 16 (50) 16 (50) 16 (50)
BRAFV600E (n,%) 7 (22) 12 (38) 0.072 7 (22) 12 (38) 0.072

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Age (years) 37±11.78 44.88±14.53 0.070 40.25±13.10 39.63±15.17 0.450
Female sex 14 (44) 14 (44) 1 14 (44) 14 (44) 1
Tumor size (cm) 2.84±1.59 1.79±0.86 0.027 2.45±1.41 2.18±1.35 0.450

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Histological variants of PTC

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Classic 7 (22) 11 (34) 0.104 5 (16) 13 (41) 0.029
Follicular 8 (25) 3 (9) 0.104 9 (28) 2 (6) 0.029
Microcarcinoma 0 (0) 2 (6) 0.104 1 (3) 1 (3) 0.029

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Warthin-like 1 (3) 0 (0) 0.104 1 (3) 0 (0) 0.029
Multifocality 3 (9) 5 (16) 0.414 3 (9) 5 (16) 0.414

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Minimum local invasion 8 (25) 14 (44) 0.022 9 (28) 13 (41) 0.127

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Lymph node metastasis 5 (16) 3 (9) 0.014 1 (3) 7 (22) 0.014
AJCC/TNM stage
I-II 16 (50) 9 (28) 0.003 14 (44) 11 (34) 0.200
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III 0 (0) 7 (22) 0.003 2 (6) 5 (16) 0.200
Risk of recurrence
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Low 8 (25) 1 (3) 0.006 8 (25) 1 (3) 0.006

Intermediate 8 (25) 15 (47) 0.006 8 (25) 15 (47) 0.006
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Table 3. Frequency of MLH1 and XPD proteins levels and its correlation with PTC
clinicopathological features.
Number
Variable MLH1 XPD
(%)
Strong Weak P value Strong Weak P value
Total cases 32 19 (59) 13 (41) 8 (25) 24 (75)
Age (years)
≤45 22 (69) 13 (41) 9 (28) 5 (16) 17 (53)
0.961 0.660
>45 10 (31) 6 (19) 4 (12.5) 3 (9) 7 (22)

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Gender
Female 28 (87.5) 18 (56) 10 (31) 8 (25) 20 (62.5)
0.135 0.217
Male 4 (12.5) 1 (3) 3 (9) 0 (0) 4 (12.5)

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Tumor size (cm)
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≤2 19 (59) 13 (41) 6 (19) 15 (47)
(12.5)

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0.208 0.533
4
>2 13 (41) 6 (19) 7 (22) 9 (28)
(12.5)
Histological variants of PTC

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Classic 18 (56) 14 () 4 (12.5) 3 (9) 15 (47)
4
Follicular 11 (34) 4 (12.5) 7 (22) 7 (22)
(12.5)
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0.036 0.489
Microcarcinoma 2 (6) 0 (0) 2 (6) 1 (3) 1 (3)
Warthin-like 1 (3) 1 () 0 (0) 0 (0) 1 (3)
Multifocality
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Present 8 (25) 7 (22) 1 (3) 1 (3) 7 (22)

0.061 0.346
Absent 24 (75) 12 (37.5) 12 (37.5) 7 (22) 17 (53)
Minimum local invasion
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Present 22 (69) 13 (41) 9 (28) 6 (19) 16 (50)

0.961 0.660
Absent 10 (31) 6 (19) 4 (12.5) 2 (6) 8 (25)
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V600E
BRAF mutation
4
Present 19 (59) 11 (34) 8 (25) (12.5) 15 (47)
0.837 0.533
4
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Absent 13 (41) 8 (25) 5 (16) (12.5) 9 (28)

Lymph node metastasis
Present 8 (25) 7 (22) 1 (3) 0 (0) 8 (25)
0.061 0.059
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Absent 24 (75) 12 (37.5) 12 (37.5) 8 (25) 16 (50)

AJCC/TNM stage
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I-II 25 (78) 16 (50) 9 (28) 6 (19) 19 (59)

0.314 0.805
III 7 (22) 3 (9) 4 (12.5) 2 (6) 5 (16)
Risk of recurrence
Low 9 (28) 4 (12.5) 5 (16) 3 (9) 6 (19)
0.282 0.496
Intermediate 23 (72) 15 (47) 8 (25) 5 (16) 18 (56)
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Table 4. Frequency of MGMT and XRCC2 proteins levels and its correlation with
PTC clinicopathological features.
Number
Variable MGMT XRCC2
(%)
Strong Weak P value Strong Weak P value
Total cases 32 23 (72) 9 (28) 10 (31) 22 (69)
Age (years)
≤45 22 (69) 17 (53) 5 (16) 6 (19) 16 (50)
0.314 0.472
>45 10 (31) 6 (19) 4 (12.5) 4 (12.5) 6 (19)

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Gender
Female 28 (87.5) 20 (63) 8 (25) 8 (25) 20 (63)
0.882 0.387
Male 4 (12.5) 3 (9) 1 (3) 2 (6) 2 (6)

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Tumor size (cm)
≤2 19 (59) 14 (44) 5 (16) 6 (19) 13 (41)
0.783 0.961

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>2 13 (41) 9 (28) 4 (12.5) 4 (12.5) 9 (28)
Histological variants of PTC
Classic 18 (56) 12 (38) 6 (19) 7 (22) 11 (34)
Follicular 11 (34) 9 (28) 2 (6) 2 (6) 9 (28)

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0.249 0.543
Microcarcinoma 2 (6) 2 (6) 0 (0) 1 (3) 1 (3)
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Warthin-like 1 (3) 0 (0) 1 (3) 0 (0) 1 (3)
Multifocality
Present 8 (25) 7 (22) 1 (3) 1 (3) 7 (22)
0.256 0.186
Absent 24 (75) 16 (50) 8 (25)
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9 (28) 15 (47)
Minimum local invasion
Present 22 (69) 16 (50) 6 (19) 4 (12.5) 18 (56)
0.874 0.018
D

Absent 10 (31) 7 (22) 3 (9) 6 (19) 4 (12.5)

V600E
BRAF mutation
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Present 19 (59) 13 (41) 6 (19) 6 (19) 13 (41)

0.599 0.961
Absent 13 (41) 10 (31) 3 (9) 4 (12.5) 9 (28)
Lymph node metastasis
Present 8 (25) 5 (16) 3 (9) 4 (12.5) 4 (12.5)
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0.496 0.186
Absent 24 (75) 18 (56) 6 (19) 6 (19) 18 (56)
AJCC/TNM stage
I-II 25 (78) 18 (56) 7 (22) 8 (25) 17 (53)
C

0.976 0.863
III 7 (22) 5 (16) 2 (6) 2 (6) 5 (16)
Risk of recurrence
AC

Low 9 (28) 6 (19) 3 (9) 4 (12.5) 5 (16)

0.682 0.314
Intermediate 23 (72) 17 (53) 6 (19) 6 (19) 17 (53)
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