Michela Perrino

The tight relationship between papillary thyroid cancer, autoimmunity and inflammation: clinical and molecular studies

Objective  The recent concept that oncogenes responsible for thyroid neoplastic transformation are able to elicit an inflammatory protumourigenic microenvironment raises interest in further studies on papillary thyroid cancer (PTC) associated with thyroid autoimmunity.

Clinical and molecular features of differentiated thyroid cancer diagnosed during pregnancy

OBJECTIVE Pregnancy represents a favorable condition for the development of thyroid nodules, likely due to the secretion of hormones with stimulatory activity. In particular, differentiated thyroid cancer (DTC) represents the second most frequent tumor among those diagnosed during pregnancy. However, few and discordant data are available about the impact of pregnancy on tumor outcome. METHODS A total of 123 women with DTC were divided into three groups according to the timing of tumor diagnosis (group 1, at least 1 year after the delivery; group 2, during pregnancy or in the first year after delivery; and group 3, before pregnancy or nulliparity) and evaluated according to the international guidelines. Furthermore, immunohistochemical studies of estrogen receptor alpha (ERalpha) were performed in 38 papillary thyroid cancer tissues from the three groups. RESULTS Thyroid cancer diagnosed during pregnancy was associated with a poorer prognosis compared to tumors developed in nongravidic periods (P<0.0001). Accordingly, at the stepwise logistic regression analysis, the diagnosis of DTC during pregnancy or in the first year post partum was the most significant indicator of persistent disease (P=0.001). Interestingly, ERalpha expression significantly differed among tumors of the three groups, being detected in 31% of group 1, in 87.5% of group 2, and in 0% of group 3 (P=0.01). CONCLUSIONS Present data indicate that pregnancy has a negative impact on the outcome of thyroid cancer. The presence of ERalpha in the majority of tumors diagnosed during pregnancy indicates that the poorer outcome of these cases could be related to the estrogen-mediated growth stimulus.

Comparison of Calcium and Pentagastrin Tests for the Diagnosis and Follow-Up of Medullary Thyroid Cancer

CONTEXT The evaluation of basal calcitonin (bCT) and stimulated calcitonin (sCT) can be used for the diagnosis and follow-up of medullary thyroid cancer (MTC). OBJECTIVE The aim of this study was to evaluate the reliability of high-calcium (Ca) test and to identify gender-specific thresholds for MTC diagnosis. PATIENTS Patients with MTC in remission (n=24) or in persistence (n=18), RET gene mutations carriers (n=14), patients with nodular goiter (n=69), and healthy volunteers (n=16) were submitted to pentagastrin and Ca (25 mg/kg) tests. RESULTS In all groups, the levels of calcitonin (CT) stimulated by either pentagastrin or Ca were significantly correlated. The prevalence of both C-cell hyperplasia (CCH) and MTC in women and men paralleled the increasing basal and peak CT levels in a gender-specific manner. Receiver operating characteristic plot analyses showed that the best levels of bCT to separate normal and CCH cases from MTC patients were above 18.7 pg/ml in females and above 68 pg/ml in males. Furthermore, Ca sCT above 184 pg/ml in females and above 1620 pg/ml in males had the highest accuracy to distinguish normal and CCH cases from patients with MTC. At the C-cell immunohistochemical examination, Ca sCT below 50 pg/ml corresponded to a mean number of 30 cells per 10 fields, whereas higher sCT associated with a mean number of 400 cells per 10 fields, often displaying a diffuse and nodular distribution pattern. CONCLUSIONS High-dose Ca test is a potent and well-tolerated procedure that can be applied worldwide at a low cost. Reference ranges for Ca sCT levels in different groups of patients and CT thresholds to diagnose CCH/MTC have been identified.

Telomerase in differentiated thyroid cancer: Promoter mutations, expression and localization

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.

Outcome predictors and impact of central node dissection and radiometabolic treatments in papillary thyroid cancers ≤2 cm

The incidence of papillary thyroid cancer (PTC) is rapidly growing, the recorded increase being mainly related to tumors < or =2 cm. The re-classification of tumors >1 and < or =2 cm limited to the thyroid from the T2 to the T1 category triggered some concerns about their best management. In order to identify possible predictors of disease outcome, several clinico-pathological features were analyzed by uni- and multivariate analyses in a retrospective consecutive series of 251 PTCs < or =2 cm. Moreover, since 37% of cases were submitted to prophylactic central compartment node dissection (CLND, VI-VII levels) and radioiodine ablation was performed only when the tumor had an extrathyroidal extension, the impact of these therapeutic tools on the final outcome was evaluated. Among all outcome predictors analyzed, only lymph node metastases and extracapsular invasion were strongly associated with persistence/recurrence. It is worth noting that neither age nor tumor size was a significant indicator of the outcome. Interestingly, as far as the therapeutic interventions are concerned, CLND was strongly associated with remission, whereas radioiodine ablation did not influence the outcome. In conclusion, present results confirm the prognostic influence of node metastases and extra-thyroidal invasion, indicating the need for aggressive treatment in tumors extending beyond the capsule. On the contrary, all pT1N0 tumors, regardless of the diameter, the number of intrathyroidal foci, and the age can be effectively treated only by surgery. The major impact of prophylactic CLND on prognosis suggests to routinely associate it to total thyroidectomy in cases with a preoperative diagnosis of malignancy.

Fetal cell microchimerism in papillary thyroid cancer: studies in peripheral blood and tissues

Fetal cell microchimerism (FCM) is defined as the persistence, for decades after pregnancy, of fetal cells in maternal organs and circulation without any apparent rejection. We recently reported evidence, in papillary thyroid cancer (PTC) tissues, supporting a possible role of FCM in tumor damage and repair. To extend those data at the peripheral level, 106 women with a previous male pregnancy, comprising 57 with PTC and 49 healthy controls were enrolled. The presence of circulating male DNA was assessed by the amplification of the Y chromosome‐specific gene SRY, with a sensitivity of 1 male cell per 1 million female cells. Moreover, to compare the microchimeric status in blood and in tumors, the neoplastic tissues of 19 women were studied. At the blood level, a significantly lower frequency of FCM was found in parous women with PTC with respect to controls (49.1% vs. 77.6%; p = 0.002). By PCR, male DNA was identified in the tumor tissues of 6 patients, and FISH analyses confirmed the presence of microchimeric cells (range 2.1–6.9 cells/section). In some patients, FCM was negative in the blood, whereas microchimeric cells were identified in the tumor. In conclusion, the prevalence of FCM in peripheral blood was found to be significantly lower in patients than in healthy controls. The presence of microchimeric cells in the tumors, but not at the peripheral level, supports the hypothesis that fetal cells could reside in maternal niches and could be recruited to diseased areas, where they could differentiate to regenerate damaged tissues.

Refining Calcium Test for the Diagnosis of Medullary Thyroid Cancer: Cutoffs, Procedures, and Safety

CONTEXT Calcitonin (CT) measurement is crucial to the early diagnosis and the follow-up of medullary thyroid cancer (MTC). If the evaluation of stimulated CT levels is required, a provocative test can be performed, being the high-dose Ca test recently reintroduced in clinical practice. OBJECTIVE Our objective was to identify gender-specific thresholds for MTC diagnosis in a large series of patients who underwent the Ca test. PATIENTS AND METHODS A total of 91 patients (49 females and 42 males) underwent the Ca test (calcium gluconate, 25 mg/kg) before thyroidectomy and both basal CT (bCT) and stimulated CT (sCT) were compared with histological results by receiver operating characteristic plot analyses. To evaluate possible side effects of Ca administration, cardiac function has been extensively studied. RESULTS bCT levels were found to harbor the same accuracy as sCT in the preoperative diagnosis of MTC. The best Ca thresholds for the identification of MTC were >26 and >68 for bCT and >79 and >544 pg/mL for sCT in females and males, respectively. The high tolerability and safety of the Ca test was demonstrated and advice offered to be followed before and during the test. CONCLUSIONS Gender-specific bCT and sCT cutoffs for the identification of C-cell hyperplasia and/or MTC have been defined. The bCT and sCT were found to have a similar accuracy, indicating that serum CT assays with improved functional sensitivity may likely decrease the relevance of the stimulation test in several conditions. Finally, systematic cardiac monitoring confirms the safety of the Ca test.

Sorafenib-Induced Destructive Thyroiditis

Sorafenib (Nexavar; Bayer Pharmaceuticals Group, Montville, NJ) is an orally active tyrosine-kinase inhibitor (TKI) whose spectrum of inhibition includes Raf kinase; vascular endothelial growth factor receptors 1, 2, and 3; platelet-derived growth factor receptor b; c-Kit protein; and RET receptor tyrosine kinases. It is used to treat advanced renal cell carcinoma and hepatocellular carcinoma (HCC) (1,2). The most common adverse events are diarrhea, rash, fatigue, hand–foot skin reactions, and hypertension. High prevalence of thyroid dysfunction, mostly hypothyroidism, occurs with sunitinib (Sutent; Pfizer Ltd., Sandwich, United Kingdom), a TKI with a similar mechanism of action. In the absence of data on sorafenib, we started routinely analyzing thyroid function during sorafenib. Here we report the first case of overt thyrotoxicosis caused by sorafenib-induced destructive thyroiditis. A 60-year-old Korean man with chronic hepatitis B virus (HBV) infection underwent liver surgery to remove a 3.5-cm nodule of HCC with microscopic vessel invasion. He was initially treated with lamivudine and adefovir dipivoxil, with sustained virological response (HBV-DNA< 12 UI/mL). Three years later he presented with multiple liver and peritoneal tumors consistent with second primary HCC. Sorafenib, 400 mg twice daily, was started after freezing a baseline serum sample. There was no history of thyroid disease, autoimmune disease, or medications that affect thyroid function, including interferons. After 4 weeks he developed fatigue, hand–foot skin reaction, dysphonia, and moderate weight loss. He was a tired-appearing man but with no apparent distress, normal, dry skin, and erythrodysesthesia of the palmar and plantar surfaces. Thyroid function tests, which were normal at baseline, showed thyrotoxicosis (Supplemental Fig. S1, available online at www.liebertonline.com). Urine iodine was normal (195mg/day; normal range, 150–300). Serum thyroglobulin was 39mg/L prior sorafenib treatment but 399mg/L after 1 month, suggesting destructive thyroiditis. This was confirmed by an ultrasonography scan showing a diffusely hypoechoic gland of normal volume (Supplemental Fig. S2A, available online at www.liebertonline.com). An echocolor Doppler showed reduced vascularization (Supplemental Fig. S2B, available online at www.liebertonline.com), and 99mTc pertechnetate thyroid scintigraphy demonstrated markedly decreased uptake of radioiodine (0.2%; normal range, 0.5%–4%). Serum antibodies against thyroglobulin, thyroid peroxidase, and thyrotropin (TSH) were negative at baseline and at month 1. Treatment with 25 mg/day of prednisone þ 40 mg/day of propanol was adopted and the original dosing of 800 mg/day of sorafenib could be maintained with a partial tumor response at month 4 of therapy. When prednisone was tapered down every 10 days over 12 weeks serum TSH remained below normal and free thyroxine levels remained elevated, but there were no symptoms and/or signs of thyrotoxicosis such as heat intolerance, tachycardia, or anxiety. Free triiodothyronine levels normalized at week 8. At week 15, TSH and free thyroxine levels normalized, and prednisone and propanolol were withdrawn. At week 18 TSH rose to the hypothyroid range with a plateau level of about 10 mU/L, with normal free thyroid hormones and no hypothyroid symptoms (Supplemental Fig. S1). We reported this adverse event to the Italian pharmacovigilance center in November 2009 (number 107996). While the clinical benefit of sorafenib is undisputed, its administration is complicated by a significant rate of mild to moderate adverse events. Hypothyroidism has been reported in 18% of patients treated with sorafenib for renal cell carcinoma with a median time of appearance of 1.8 months (3). Consistently, in our series of 38 consecutive patients with HCC treated with sorafenib, 5 (13%) developed a subclinical hypothyroidism (TSH levels, 7.41mIU/mL; range, 6.38–8.94mIU/ mL) (data not shown). Moreover, here we report the first description of sorafenib-induced thyrotoxicosis. Surprisingly, sorafenib-associated thyroid dysfunction was not reported in two registration trials in patients with advanced HCC (2,4). We speculate that sorafenib-induced hypothyroidism could have the same basis as sunitinib-induced hypothyroidism, including reduced synthesis of thyroid hormones related to inhibition of thyroid peroxidase activity, destructive thyroiditis, and inhibition of thyroid uptake of iodine (5). Nevertheless, thyroid toxicity occurs more frequently in patients receiving the TKI sunitinib (up to 85% of cases) than in those taking sorafenib, possibly indicating different pathogeneses. In contrast, sunitinib-induced mild thyrotoxicosis with undetectable TSH levels and normal free thyroid hormones is less frequent, and overt thyrotoxicosis unusual (6). Thus, thyroid dysfunction is a class-specific adverse event of TKIs. The picture of overt hyperthyroidism after short-term

The optimal range of RET mutations to be tested: European comments to the guidelines of the American Thyroid Association

In the 9th ETA-CRN Meeting (September 2009, Lisbon) some recommendations from the American Thyroid Association (ATA) guidelines for the management of medullary thyroid cancer (MTC) were discussed by an European Panel of Experts (EPE). Among the 12 ATA recommendations related to hereditary MTC and to the optimal range of RET mutations to be tested (recommendations 1-5 and 9-15), 7 were shared and 5 were not shared by the EPE. In the present paper, the related comments and suggestions will be reported and discussed.

Papillary Thyroid Carcinoma and Inflammation

The relationship between cancer and inflammation is well known since 1863 when Rudolf Virchow, following the observation of leukocytes in neoplastic tissues, hypothesized that chronic inflammation could contribute to the tumorigenic process. In the following decades, several lines of evidence suggested a strong association between chronic inflammation and increased susceptibility to neoplastic transformation and cancer development. It was estimated that up to 20% of all tumors arise from conditions of persistent inflammation such as chronic infections or autoimmune diseases. Indeed, the associations are well known between cervical cancer and papilloma virus, gastric cancer and Helicobacter pylori induced gastritis, esophageal adenocarcinoma and Barretts metaplasia, hepatocellular carcinoma, hepatitis B and C viral infections, and many others. Some of the mechanisms forming the basis of the relationship between inflammation and tumor have been recently elucidated. The inflammatory microenvironment of neoplastic tissues is characterized by the presence of host leukocytes both in the supporting stroma and among the tumor cells, with macrophages, dendritic cells, mast cells, and T cells being differentially distributed (Balkwill and Mantovani, 2001). Several cytokines (TNF, IL-1, IL-6) and chemokines that are produced by the tumor cells and by leukocytes and platelets associated with the tumor have been found to be able to maintain the invasive phenotype (Coussens and Werb, 2002). Tumor-associated macrophages (TAMs) are a major component of the leukocyte infiltrate, initially recruited by inflammatory chemokines (e.g., CCL2) and then sustained by cytokines present in the tumor microenvironment (e.g., CSFs, VEGF-A). In response to cytokines such as TGF-β, IL-10, and M-CSF, TAMs promote tumor proliferation and progression and stroma deposition and, indeed, the density of TAMs is increased in advanced thyroid cancers (Ryder et al., 2008). As far as papillary thyroid cancer (PTC) is concerned, this tumor is frequently associated with autoimmune thyroid diseases, Graves’ disease, and Hashimotos thyroiditis. The frequency of association is extremely variable in the series from different countries, 0–9% for Graves’ and 9–58% for Hashimotos (Figure ​(Figure1).1). It is still debated whether association with an autoimmune disorder could influence the prognosis of PTC. Indeed a worse prognosis was reported in few series (Ozaki et al., 1990; Pellegriti et al., 1998), while the majority of the studies showed either a protective effect of thyroid autoimmunity (Matsubasyashi et al., 1995; Loh et al., 1999; Gupta et al., 2001) or a similar behavior between cancer with and without associated thyroiditis (Yano et al., 2007). These discrepancies can be due to either the low number of patients examined in those studies, the lack of a control group, the existence of different genetic and epidemiological backgrounds, or the use of inappropriate criteria to define remission or persistence/relapse. We recently produced data extending the knowledge about the tight relationships among thyroiditis and thyroid cancer. In particular, the clinical and molecular features, and the expression of inflammation-related genes, were investigated in a large series of PTCs divided in two groups according to the association or not of the tumor with thyroiditis (Muzza et al., 2010). Interestingly, no significant differences between the two groups were found, as far as age at diagnosis, gender distribution, TNM staging, histological variants, and outcome are concerned, suggesting that the association with an autoimmune thyroid process does not modify either the presentation or the clinical behavior of PTC. A crucial finding of the last few years concerns the genetic background of PTCs, since the concept has emerged that the inflammatory protumourigenic microenvironment of this cancer is elicited by the oncogenes responsible for thyroid neoplastic transformation (such as RET/PTC, BRAFV600E, and RASG12V; Borrello et al., 2005, 2008; Melillo et al., 2005; Mantovani et al., 2008). In particular, we recently demonstrated that the RET/PTC1 oncogene activates a transcriptional proinflammatory program in normal human primary thyrocytes (Borrello et al., 2005). Moreover, gene expression studies in cellular systems showed that not only RET/PTC but also RAS and BRAF proteins, all belonging to the RET–PTC/RAS/BRAF/ERK pathway, are able to induce the up-regulation of chemokines, which in turn could contribute to neoplastic proliferation, survival, and migration (Melillo et al., 2005). Consistently, other Authors demonstrated that RET/PTC3-thyrocytes express high levels of proinflammatory cytokines (Russel et al., 2003) and proteins involved in the immune response (Puxeddu et al., 2005). These data are well in agreement with our recent study which firstly showed that PTCs harbor a different genetic background according to the association or not with thyroiditis (Muzza et al., 2010). In particular, RET/PTC was more represented in patients with PTC and autoimmunity, while BRAFV600E was significantly more frequent in patients with PTC alone. Moreover, we showed that the expression of genes encoding three inflammation-related genes (CCL20, CXCL8, and l-selectin) was enhanced either in BRAFV600E or in RET/PTC tumors, compared with normal samples. Interestingly, non-neoplastic tissues with thyroiditis displayed the same levels of expression of CCL20 and CXCL8 compared to normal samples, suggesting that these inflammatory molecules could be associated with tumor-related inflammation, and not with the autoimmune process. Figure 1 Worldwide prevalence of papillary thyroid cancer in patients with Graves’ disease (Graves) and Hashimotos thyroiditis (Hashi), corresponding to the sum of the data reported to date in the literature. References: Australia (Graves: Hales et al., ... In conclusion, recent studies opened a new and extremely attractive scenario on the “connection” between thyroid autoimmunity, inflammation, and cancer. The interest is linked not only to the possibility of better understanding the communication between abnormally growing cells and their microenvironment, but also to the chance to pharmacologically interfere with such pro-tumor interactions.