Thyroid autoimmunity and risk of malignancy in thyroid nodules submitted to fine-needle aspiration cytology

ORIGINAL ARTICLE Thyroid autoimmunity and risk of malignancy in thyroid nodules submitted to fine-needle aspiration cytology Giorgio Grani, MD, Anna Calvanese, MD, Giovanni Carbotta, MD, Mimma D Alessandri, MD, Angela Nesca, MD, Marta Bianchini, MD, Marianna Del Sordo, MD, Martina Vitale, MD, Angela Fumarola, MD * Department of Experimental Medicine, Unit of Endocrinology, Sapienza Universita di Roma, Rome, Italy. Accepted 20 December 2013 Published online 3 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23587 ABSTRACT: Background. Whether the risk of cancer is increased in with chronic autoimmune thyroiditis is a controversial issue. Methods. Between May 2005 and October 2012, 3777 fine-needle aspiration cytologies (FNACs) were performed on 2562. Serum FT4, thyroid-stimulating hormone (TSH), anti-thyroglobulin antibody (TgAb), and anti-thyroperoxidase antibody (TPOAb) were determined. Results. Patients with suspicious cytology were younger and presented smaller maximum lesion diameter. In with TgAb positivity, suspicious cytology was detected more frequently (9.4%) than without TgAb (5.7%; p 5.04). No significant difference was recorded between benign and suspicious cytology in the positive TPOAb rate. Risk factors for suspicious cytology were younger age (odds ratio [OR], 0.94), smaller maximum diameter (0.95), single lesion (1.85), microcalcifications (3.45), and TgAb (1.74). Mixed solid/fluid content resulted as being a protective factor (0.34). According to multivariate logistic regression analysis, age, mixed content, and microcalcification confirmed significance. Conclusion. Thyroid nodule malignancy in with Hashimoto thyroiditis is not more frequent than in without thyroiditis. VC 2014 Wiley Periodicals, Inc. Head Neck 37: 260 264, 2015 KEY WORDS: Hashimoto disease, thyroid neoplasms, biopsy, fineneedle aspiration cytology, autoimmunity, cytology INTRODUCTION The association between Hashimoto thyroiditis and differentiated thyroid cancer (DTC) was suggested for the first time in 1955 by Dailey et al 1 and was later confirmed by Okayasu et al 2 in 1995. The causal relationship has yet to be clarified, although the expression of RET/PTC1 and RET/PTC3 rearrangements have been described in with Hashimoto thyroiditis, which are well-known milestones in the genetic pathway of papillary thyroid carcinoma (PTC). 3 Indeed, the expression of other PTC markers, such as cytokeratin 19 4 and p63, 5 has been described in chronic autoimmune thyroiditis thyroid specimens. Moreover, chronic autoimmune thyroiditis, the leading cause of primary hypothyroidism, determines the rise in serum thyroid-stimulating hormone (TSH) levels that may hypothetically stimulate tumor growth. 6 8 The association has been analyzed by several authors, but the issue is still the object of controversy in the literature. 9,10 Although some authors report a higher risk of PTC in affected by chronic autoimmune thyroiditis, others, on the other hand, do not confirm this *Corresponding author: A. Fumarola, Department of Experimental Medicine, Sapienza Universita di Roma, V. le Regina Elena, 324, 00161 Rome, Italy. E-mail: angela.fumarola@uniroma1.it This work was presented at the 15th International and 14th European Congress of Endocrinology, Florence, Italy, May 5 9, 2012. finding. This disagreement depends both on the type and design of the studies. In fact, the association between chronic lymphocytic thyroiditis and thyroid cancer has been shown to be statistically significant in most of the retrospective studies conducted on thyroid specimens after total thyroidectomy for various indications. 11 13 The main limitations of this kind of study are the difficulty in histological differentiation of a peritumoral lymphocytic infiltrate from a real lymphocytic thyroiditis and a possible selection bias, as only who underwent total thyroidectomy have been evaluated. Based on this, other studies have been conducted from a different point of view. Boi et al 14 have evaluated the cytological features of thyroid nodules subjected to fine-needle aspiration cytology (FNAC). The cytological analysis on 590 individual nodules revealed a relationship between the Tir 4 cytological result (predictive in 90% of PTC) and at least 1 positive anti-thyroid autoantibody. The main limitation of this study was that the positivity of just 1 autoantibody is not necessarily an indication of thyroiditis diagnosis. In another study, Kim et al 15 have examined the 2 main thyroid autoantibodies, anti-thyroglobulin autoantibodies (TgAbs) and anti-thyroperoxidase autoantibodies (TPOAbs), independently, in who underwent FNAC. No association between cancer and high levels of TPOAb was found, but only between cancer and the presence of TgAbs, which are less specific markers and not necessarily diagnostic of Hashimoto thyroiditis. 260 HEAD & NECK DOI 10.1002/HED FEBRUARY 2015

THYROID AUTOIMMUNITY AND RISK OF CANCER FIGURE 1. Schematic diagram of the study sample. It is common knowledge that the prevalence of high TgAb in with DTC is higher than in the general population. Antibody levels decrease within 3 years after thyroidectomy in a patient free from disease, whereas levels of both thyroglobulin (Tg) and TgAb remain high in case of relapse or persistence of disease. This is probably because of the fact that thyroid carcinoma cells release Tg and stimulate the immunologic response against it. An interesting prospective study has been conducted by Anil et al 16 on with positive antibodies in which the prevalence of malignancy (evaluated via FNAC) was 1%, whereas in the control population, it was 2.7%, a nonsignificant difference. MATERIALS AND METHODS Between May 2005 and October 2012, 3777 consecutive ultrasonography-guided FNACs were performed at the Thyroid Center of Sapienza University of Rome. Considering only the last aspiration, 2962 suspicious nodules of 2562 were sampled (Figure 1). Serumfree thyroxine (FT4), free triiodothyronine (FT3), TSH, calcitonin, TgAb, and TPOAb were determined at the time of FNAC. Anti-TPO and anti-tg antibodies were measured using radioimmunoassays and were considered positive if found to be above the cutoff point set by the laboratory (>50 U/mL). Definition of Hashimoto thyroiditis Hashimoto thyroiditis was clinically diagnosed in showing a heterogeneous thyroid parenchyma by ultrasonography and high TPOAb or TgAb. However, the final diagnosis is known to be based on histological data: extensive lymphocytic infiltration of the stroma, eosinophilic change of the follicular epithelium (H urthle or Askanazy cells), and interstitial fibrosis. There is no international consensus on criteria used to diagnose Hashimoto thyroiditis in not undergoing thyroidectomy. For this reason, major features of lymphocytic thyroiditis (TgAb, TPOAb, and TSH) were individually analyzed. Selection In all, at least 1 discrete nodular lesion of the thyroid or multinodular goiter had been detected. These were then referred to our institution to undergo FNAC, selected on the basis of even a single ultrasonography feature (irregular margins, microcalcifications, internal vascularization, or marked hypoechogenicity) or clinical factor suggestive of malignancy, such as history of neck irradiation, family history of thyroid carcinoma, male sex, and/or growing nodule. Functional status Thyroid functional status was evaluated according to serum TSH levels. TSH levels were considered only in 1708 FNACs performed in without substitutive or suppressive therapy with levothyroxine. Ultrasound-guided FNAC was performed by trained endocrinologists using a Toshiba Aplio XV device (Toshiba Medical Systems Europe B.V, Zoetermeer, The Netherlands) equipped with a linear high-frequency transducer, referring to an internal examination protocol to reduce variability. Ultrasound-guided FNAC was performed via aspiration using 23 25 gauge needles attached to a 10- ml syringe. The first smear was air-dried and stained using the May Grunwald Giemsa method. The remaining smears were fixed with Bio-fix (Bioptica, Milan, Italy) and stained with hematoxylin-eosin. Cytology results were reported in 5 categories, as follows, in accord with the British Thyroid Association Guidelines 17 and the Thyroid Cytology Italian Consensus SIAPEC-IAP 18 : (1) nondiagnostic, (2) benign, (3) indeterminate, (4) probably malignant, and (5) positive for malignant cells. Smears were reviewed by at least 2 skilled pathologists. Seven samples suspicious for medullary thyroid cancer were excluded from further evaluation. Inadequate samples (characterized by <6 groups of thyrocytes containing at least 10 cells each) were reported in 826 FNACs and excluded from further evaluation. Patients with benign nodules underwent clinical follow-up (mean, 27.76 months; range, 0 92 months). Results falling into the fourth and fifth classes were considered suspicious for thyroid cancer. Among the 92 included in these classes, 46 underwent surgery with histological examination, which always confirmed malignancy (1 follicular thyroid cancer, 42 PTC, and 3 follicular variants of PTC). The remaining 46 refused surgery or HEAD & NECK DOI 10.1002/HED FEBRUARY 2015 261

GRANI ET AL. TABLE 1. Clinical characteristics and results of univariate and multivariate logistic regression analysis. Clinical characteristics Univariate logistic regression analysis Multivariate logistic regression analysis* Benign cytology Suspicious cytology OR (95% CI) OR (95% CI) No. of 1889 100 Sex, female 1617 87.77 1.13 (0.62 2.04).70 Age, y, mean 6 SD 55.89 6 13.06 45.07 6 16.17 <.001 0.94 (0.93 0.96) <.001 0.94 (0.92 0.95) <.001 TSH, miu/l, median and 1.00 (1 2) 1.66 (1 2).002 1.00 (0.99 1.01).94 interquartile range Maximum nodule diameter, 15.70 6 8.12 13.09 6 7.13.002 0.95(0.92 0.98).002 0.99 (0.96 1.03).80 mm, mean 6 SD Single nodule, number 276 24.014 1.85 (1.15 2.97).012 1.36 (0.76 2.45).30 Mixed, solid, and cystic 644 15 <.001 0.34 (0.19 0.60) <.001 0.41 (0.21 20.80).009 component nodule number Microcalcifications, number 465 53 <.001 3.45 (2.30 5.19) <.001 4.41 (2.68 7.26) <.001 TgAb, number 230 24.043 1.74 (1.05 2.88).03 0.98 (0.41 2.38).97 TPOAb positive, number 366 23.36 1.25 (0.77 2.02).37 TgAb and TPOAb positive, 134 11.33 1.62 (0.84 3.10).15 number Either TgAb or TPOAb positive, number* 302 31.017 2.36 (1.52 3.67) <.001 1.65 (0.72 3.75).24 Abbreviations: OR, odds ratio; CI, confidence interval; TSH, thyroid-stimulating hormone; TgAb, thyroglobulin antibody; TPOAb, thyroid peroxidase antibody. * Measured in 1177 cases. Measured in 1106 cases. Measured in 1209 cases. Measured in 1954 cases. Measured in 1177 cases. Note: Multivariate analysis included only factors that proved significant in univariate analysis. Fine-needle aspiration cytology (FNAC) reports of inadequate sample (826), indeterminate cytology (141), or suspicious for medullary thyroid cancer (6) were excluded. were otherwise lost to follow-up. Indeterminate FNAC reports (141) were excluded from logistic regression analysis (Figure 1). Statistical analysis Data are expressed as mean 6 SD. Categorical variables were compared using the Fisher exact test or the Pearson chi-square test. Continuous variables were compared using the unpaired t test or Mann Whitney U test when appropriate. Univariate and multivariate logistic regression analyses were performed to evaluate the association between suspicious cytology and risk factors, such as age, sex, single nodule, maximum nodule diameter, and nodule estimated volume. All tests used a 2-sided a of 0.05. Analyses were performed using IBM SPSS Statistics for Windows 20.0 software (IBM, Armonk, NY). All gave written informed consent. RESULTS The were aged 55.88 6 13.86 years (mean 6 SD) and all resided in Central Italy, an area of mild to moderate iodine deficiency. Male and female were not significantly different for age: 429 men were aged 56.95 6 14.16 years, whereas 2133 women were aged 55.66 6 13.79 years (p 5 ns). TPOAb were recorded for 2510 and were found to be positive in 477 cases (19.0%), whereas TgAb were recorded in 1541 and were positive in 291 (18.9%). No significant difference was recorded between with benign and suspicious cytology in sex ratio, in the rate of positive TPOAb or in the rate of both autoantibody positives (Table 1). Patients with suspicious cytology were younger (45.07 6 16.17 vs 55.89 6 13.06 years; p <.001), and presented smaller maximum lesion diameter (13.09 6 7.13 mm vs 15.70 6 8.12; p 5.002). Although these data could seem disappointing and raise concerns about patient selection, they are consistent with several recent articles. 19,20 In with TgAb positivity, suspicious cytology was detected more frequently than in without TgAb (9.4% vs 5.7%; p 5.04), independently from TSH levels. Detection of microcalcifications and single nodule was associated with a significantly greater rate of suspicious cytology. According to univariate analysis, risk factors for thyroid cancer with suspicious cytology were younger age (odds ratio [OR], 0.94; 95% confidence interval [CI], 0.93 0.96; p <.001), smaller maximum diameter (OR, 0.95; 95% CI, 0.92 0.98; p 5.002), single lesion (OR, 1.85; 95% CI, 1.15 2.97; p 5.012), presence of microcalcifications (OR, 3.45; 95% CI, 2.30 5.19; p <.001), TgAb positivity (OR, 1.74; 95% CI, 1.05 2.88; p 5.03), and a single antibody (either TgAb or TPOAb) positivity (OR, 2.36; 95% CI, 1.52 3.67; p <.001). Mixed content of the lesion (solid and cystic component) turned out to be a protective factor (OR, 0.34; 95% CI, 0.19 0.60; p <.001). All these factors were included in a multivariate logistic regression analysis model, however, only age (OR, 0.94; 95% CI, 0.92 0.95; p <.001), mixed content (OR, 0.41; 95% CI, 0.21 0.80; p 5.01), and microcalcification (OR, 4.41; 95% CI, 2.68 7.26; p <.001) confirmed statistic significance. Upon computing FNAC report classes separately 262 HEAD & NECK DOI 10.1002/HED FEBRUARY 2015

THYROID AUTOIMMUNITY AND RISK OF CANCER TABLE 2. Comparison of cytology report classes between thyroid peroxidase antibody negative and positive groups and thyroglobulin antibody negative and positive groups (Fisher test). TPOAb TgAb Cytology report Thyroid 2 1490 (88.6%) 366 (87.8%).60 901 (87.4%) 230 (85.5%).42 3 110 (6.5%) 28 (6.7%).91 74 (7.2%) 15 (5.6%).42 4 58 (3.5%) 19 (4.6%).31 42 (4.1%) 18 (6.7%).07 5 17 (1.0%) 4 (1.0%) 1.00 12 (1.2%) 6 (2.2%).24 MTC 6 (0.4%) 0 (0.0%).60 2 (0.2%) 0 (0.0%) 1.00 Total 1681 417 1031 269 Abbreviations: TPOAb, thyroid peroxidase antibody; TgAb: thyroglobulin antibody; MTC, medullary thyroid cancer. (with the exception of inadequate samples), there was no significant difference found between TPOAb positive or negative groups and between TgAb positive or negative groups (Table 2). This finding is confirmed when only nodules >1 cm in maximum diameter were considered (Table 3). DISCUSSION Several studies discussed the relationship between thyroid autoantibodies and the risk of thyroid cancer. Both TPOAb and TgAb have been widely associated with numerous illnesses, in part because they are present in up to 10% of the general female population. This issue is clinically relevant, as a careful clinical monitoring and a more aggressive clinical management of nodules is called for, should Hashimoto thyroiditis be confirmed as a risk factor for thyroid cancer. 10 A major limitation of the present study was the retrospective analysis and the lacking of histological follow-up. However, the majority of published articles evaluated this association by analyzing surgical series, and by considering TPOAb and TgAb altogether, and not individually. Histology is the only method to conclusively diagnose both thyroid cancer and chronic lymphocytic thyroiditis. However, as thyroiditis is a benign and frequent disease that usually does not require surgery, the subgroup of who require thyroidectomy is at higher risk for malignancy compared to the general population with Hashimoto thyroiditis. Thus, the study of histopathological series is also a source of relevant selection bias. Furthermore, a peritumoral lymphocytic infiltrate (focal thyroiditis) is common around DTC and could be misdiagnosed as chronic lymphocytic thyroiditis. Other sources of heterogeneity should be considered, such as different diagnostic criteria for thyroiditis and surgical procedures. 10 The present study showed that Hashimoto thyroiditis was not predictive of suspicious cytology. This is confirmed either by using TPOAb as the most specific single diagnostic marker of chronic autoimmune thyroiditis 21 or by using both autoantibody positivities as diagnostic criteria. A recent study proposed that the frequency of PTC is significantly higher in nodular thyroiditis than in nodular goiter because of increased TSH serum levels and that treatment with levothyroxine could reduce TSH levels and subsequently decrease the risk of cancer. 8 The present data could be consistent with this hypothesis because TSH was found to be marginally higher in with suspicious cytology. However, TSH is not a significant predictor of suspicious cytology, according to logistic regression analysis. Therefore, TgAb by itself can be considered (based on univariate analysis and not confirmed by multivariate analysis) a predictor of the risk of thyroid cancer, not always indicating the presence of thyroiditis. However, the estimated risk is lower (OR, 1.74) than other well-recognized risk factors, such as microcalcification (OR, 3.45) and solitary nodule (OR, 1.85). In conclusion, an isolated TgAb positivity can be considered a mild risk factor for thyroid malignancy, but TABLE 3. Comparison of cytology report classes between thyroid peroxidase antibody negative and positive groups and thyroglobulin antibody negative and positive groups, when only nodules of maximum diameter >1 cm are considered (Fisher test). TPOAb TgAb Cytology report Thyroid 2 1046 (89.2%) 252 (86.3%).18 617 (87.8%) 136 (84.0%).19 3 86 (7.3%) 26 (8.9%).39 56 (8.0%) 14 (8.6%).75 4 28 (2.4%) 10 (3.4%).31 23 (3.3%) 7 (4.3%).48 5 10 (0.9%) 4 (1.4%).50 7 (1.0%) 5 (3.1%).06 MTC 2 (0.2%) 0 (0.0%) 1.00 0 (0.0%) 0 (0.0%) - Total 1172 292 703 162 Abbreviations: TPOAb, thyroid peroxidase antibody; TgAb, thyroglobulin antibody; MTC, medullary thyroid cancer. HEAD & NECK DOI 10.1002/HED FEBRUARY 2015 263

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