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Papillomaviridae, Thyroid cancer, Papillary, Thyroid neoplasms, What&,rsquo s Known Known risk factors for the development of papillary thyroid carcinoma are exposure to ionizing radiation, high iodine intake, autoimmune thyroid diseases, and genetic predisposition. The role of parvovirus B19, herpesvirus, and polyomavirus in the pathogenesis of papillary thyroid carcinoma has been suggested. Human papillomavirus causes various cancer types. What&,rsquo s New For the first time, we demonstrated the presence of human papillomavirus DNA in thyroid tissue and its association with papillary thyroid carcinoma. IntroductionPapillary thyroid carcinoma (PTC) is the most common thyroid malignancy comprising 50-90% of the differentiated (follicular cell) thyroid carcinomas. 1, Despite many research studies, the etiology of this common thyroid malignancy remains unclear. 2, Possible risk factors for PTC are exposure to ionizing radiation, high iodine intake, autoimmune thyroid disease, and genetic predisposition. 2, The role of parvovirus B19 in the pathogenesis of PTC has also been suggested. 3, , 4, A study reported the presence of a high proportion of parvovirus B19 nucleic acids and proteins in PTC tissues. 3, In addition, a correlation between parvovirus B19 and all types of thyroid cancer has been reported. 4, A probable effect of parvovirus B19 on the activation of nuclear factor-kappa light chain enhancer of activated B cells (NF-kB ) has been indicated as a factor in the pathogenesis of PTC. 4, In contrast to previous studies, Adamson and others reported no association between parvovirus B19 and PTC. 5, A recent study, however, showed that thyroid cancer tissues harbored viral DNA or viral gene products of both the polyoma and herpes family viruses. 6, Human papillomavirus (HPV) is a non-enveloped, double-stranded DNA virus from the papillomavirus family, which has more than 170 HPV genotypes. 7, , 8, HPV is divided into two groups, based on the ability to cause neoplastic transformation, first is the benign type and the second is high-risk HPV types (HPV 16, 18, 31, 33, 35, 45, 51, 52, and 56). 9, They play an important role in cancers of the anus and genital organs. 9, A previous study has shown that persistent HPV infection can lead to precancerous lesions. 10, Recently, HPV has been implicated in head and neck carcinomas, especially oropharyngeal cancer. 7, HPV was detected in 25% of the head and neck squamous cell carcinoma cases. 11, In addition, it is suggested that HPV vaccines can have a significant health benefit in preventing oropharyngeal cancers. 11, However, it is unclear whether this virus can also play a role in other cancers of the head and neck, such as thyroid cancer. 10, , 11, Despite a relatively high prevalence of thyroid cancer and the availability of HPV vaccines for cancer prevention, studies on the role of HPV infection in thyroid cancers are scarce. The present study aimed to evaluate the association between HPV infection and PTC in comparison with benign thyroid nodules and normal thyroid tissues. In addition, the association between HPV infection and PTC severity was evaluated using tumor staging criteria and pathologic features. To the best of our knowledge, this is the first time such a study has been conducted. Materials and MethodsA retrospective cross-sectional study was conducted from May 2014 to January 2018 in hospitals affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. The study was approved by the Ethics Committee of Shiraz University of Medical Sciences (code, IR.SUMS.REC.1396 S90). Sample SizeIn the absence of any previous studies on the association between PTC and HPV, data from studies on the association between PCT and other viruses were used. The sample size was calculated (95% confidence interval, 5% significance level) using the MedCalc statistical software (Ver. 19.1.7 Med Calc software Ltd., Ostend, Belgium).The calculated sample size was 44 for each group, but it was increased to 82 PTC samples and 77 benign thyroid nodules.Tissue SamplesThyroid tissue specimens from patients diagnosed with PTC and benign thyroid nodules were collected using the consecutive sampling method. A total of 175 paraffin-embedded thyroid tissues with the diagnosis of PTC and benign thyroid nodules were collected. All thyroid tissues were re-examined by two expert thyroid pathologists to confirm the diagnosis. Medical records of the patients were evaluated to exclude samples from patients, who were immunocompromised, had a history of head and neck radiation or underwent chemotherapy. Accordingly, 16 samples from patients with Hashimoto thyroiditis or Graves&,rsquo disease were excluded from the study. Eventually, 82 PTC and 77 benign thyroid nodules (adenomatous and colloid nodules) specimens were included. In PTC specimens, samples from adjacent normal non-tumoral thyroid tissue were also taken. The data was anonymized throughout the study using irreversible encryption.Tumor Staging and Pathologic CriteriaIn accordance with the classification by the World Health Organization (WHO), 12, PTC histological types were classified into six groups, classic, encapsulated follicular, infiltrative follicular, tall cell, cribriform, and diffuse sclerosing variants. Tumor staging was determined according to the American Joint Committee on Cancer and tumor-node-metastasis staging system. 13, Pathologic features were evaluated according to the criteria specified by the College of American pathologists. 14, Sample Preparation and AnalysisTissue specimens were cut in 7 &,micro m thickness, deparaffinized in xylene (GeNet Bio, South Korea), and then washed in ethanol (GeNet Bio, South Korea). DNA extraction was performed using the PrimePrep Genomic DNA isolation kit (GeNet Bio, South Korea).All DNA samples were amplified with both primer pairs as described in table 1,. The first-round polymerase chain reaction (PCR) amplification was as follows, 12.5 &,micro L Taq 2X Master Mix (BioFact, South Korea), 0.4 &,micro m of each MY09 and MY11 primers (Metabion, Germany), 20 ng of DNA, and up to 25 &,micro L PCR-grade water. The second-round PCR (nPCR) was carried out using GP5+/6+ primers and a housekeeping gene (&,beta -actin) to assess DNA integrity with forward primer 5`-ATCATGTTGAGACCTCCAA-3`and reverse primer 5`-CATCTCTTGCTCGAAGTCCA-3`. The nPCR reaction contained 12.5 &,micro L Taq 2X Master Mix, 0.3 &,micro m of each Gp5+/6+ primers, 0.2 &,micro m of each of the forward and reverse &,beta -actin primers, 2 &,micro L of PCR product of the first-round PCR, and 8 &,micro L of the PCR-grade water. All PCR amplifications were performed on a 96-well thermal cycler (Applied Biosystems, Foster, CA, USA) using a PCR.NameSequence (5&,rsquo -3&,rsquo )Size (base pair)MY9GTCCMARRGGAWACTGATC450MY11GCMCAGGGWCTATAAYAATGGGP5+TTTGTTACTGTGGTAGATACYAC140GP6+GAAAAATAAACTGTAAATCATATTCTable 1.Human papillomavirus nested polymerase chain reaction primer sequencesPCR products were electrophoresed on a 2.5% agarose gel (BioFact, South Korea)in 1X TAE buffer (BioFact, South Korea), stained with gel Red EcoDyeTM Nucleic Acid Staining Solution (BioFact, South Korea), and visualized with a UV trans-illumination (UVItec Ltd, UK). The DNA amplification was 450 bp for the first-round PCR, 150 dp for the nPCR, and 317 dp for the &,beta -actin gene. Uterine cervix tissue of a known HPV positive specimen, earlier analyzed during nPCR, was used as a positive tissue control to evaluate the success of the amplification. Blood samples of babies suspicious of kala-azar were used as negative controls (figure 1,).Figure 1. Agarose gel electrophoresis image of common human papillomavirus DNA in a nested multiplex polymerase chain reaction in a papillary thyroid cancer specimen. The length of amplicons generated with MY09/011* primers is 450 base pairs, GP05/06* is 150 base pairs, and &,beta -actin primers 317 base pairs in size. Lane 1 is distilled water control. Distilled water in place of the DNA template was used as a negative control. Lane 2 is a negative control sample (317 base pairs &,beta -actin) for the true negative result of common human papillomavirus detection. Lanes 5-6 are positive samples for first and nested human papillomavirus polymerase chain reaction and &,beta -actin. Lanes 7 and 9 are positive samples for the nested and &,beta -actin polymerase chain reaction. Lane 8 is positive control consisted of DNA from vaginal condyloma acuminatum. Lanes 10, 11, 13 are negative samples for the first and nested human papillomavirus polymerase chain reaction. Lane 12 is DNA Ladder, 50 base pairs. *MY09/011 and GP05/06 are primers for the detection of human papillomavirus in the polymerase chain reaction. Statistical AnalysisData were analyzed using SPSS software (version 21.0, Chicago, IL, USA). HPV positive tests were compared in two groups of PTC and thyroid nodule, and PTC and adjacent normal tissue using Chi-squared and Fisher&,rsquo s exact tests. These tests were also used to evaluate the association between the qualitative pathologic features of PTC and the presence of HPV. The independent sample t test was used to compare quantitative variables such as age and tumor diameter. P values less than 0.05 were considered statistically significant.ResultsOf the 159 samples, 77 tissue specimens were benign thyroid nodules and 82 were PTC. A total of 30 samples were from male patients and 129 from female patients. The benign thyroid nodules are composed of 32 colloids (42%) and 45 (58%) adenomatous nodules ( table 2,). The prevalence of HPV PCR positivity in the PTC tissues was significantly higher than the benign thyroid nodules. Logistic regression analysis (&,ldquo enter&,rdquo method) was used to eliminate age and sex as the two probable confounding factors ( table 3,). The results showed that the prevalence of HPV positive PCR in the PTC group remained significantly higher than the benign thyroid nodules group (P=0.015). HPV was not detected in any adjacent normal non-tumoral thyroid tissue samples and the difference with the PTC group was significant (P&,lt 0.001).VariablePTC N (%)Thyroid nodule N (%)TotalP value 82 (51.5)77 (48.5)159 (100)-SexMale 21 (25.6)9 (11.6)30 (18.8)0.025*,Female61 (74.4)68 (88.3)129 (81.2)HPV+ 11 (13.4)3 (3.8)14 (8)0.034*,Age (year) 40.7&,plusmn 14.9&,sect ,46.9&,plusmn 13.3&,sect ,-0.006**,*Chi-squared test, **Independent sample t test, &,sect Mean&,plusmn standard deviation PTC, Papillary thyroid carcinoma, HPV, Human papillomavirus |