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Purpose: Distant metastasis from papillary thyroid cancer is rare, and the more frequent site is neck and bone. During follow-up after surgery new distant lesions could appear, while Tg levels remain normal. When suspicious lesions are superficial, fine needle aspiration is the first choice. In case of deeper localization, the diagnosis could need more invasive procedure, such as in case of lung nodules., Methods: We report a case of a patient with a lung lesion suspicious for metastasis from papillary thyroid cancer. The patient underwent thyroidectomy and radioiodine 10 years before and after 2 years of follow-up was submitted to right cervical lymphadenectomy for nodal recurrence. During the following 5 years Tg values on TSH suppressive treatment ranged 3.7-5.4, always with negative TgAb and negative neck US. CT scans showed the presence of a single nodule in the left lung with subpleuric localization, progressively increasing in size. A CT-guided fine needle aspiration was performed., Results: The Tg levels measured in the washout out of the Chiba's needle (386 ng/ml) provided an unequivocal demonstration of the thyroid origin of the nodule., Conclusions: Thyroglobulin measurement in the specimen obtained from CT-guided fine needle aspiration is a useful and effective tool to diagnose lung metastasis from thyroid cancer. This procedure could avoid a surgical approach that is more invasive and potentially detrimental for the patient.

Introduction: Conservative active surveillance has been proposed for low-risk papillary thyroid microcarcinoma (PTMC), defined as ≤1.0 cm and lacking clinical aggressive features, but controversy exists with accepting it as not all such PTMCs are uniformly destined for benign prognosis. This study investigated whether BRAF V600E status could further risk stratify PTMC, particularly low-risk PTMC, and can thus help with more accurate case selection for conservative management., Methods: This international multicenter study included 743 patients treated with total thyroidectomy for PTMC (584 women and 159 men), with a median age of 49 years (interquartile range [IQR], 39-59 years) and a median follow-up time of 53 months (IQR, 25-93 months)., Results: On overall analyses of all PTMCs, tumour recurrences were 6.4% (32/502) versus 10.8% (26/241) in BRAF mutation-negative versus BRAF mutation-positive patients (P = 0.041), with a hazard ratio (HR) of 2.44 (95% CI (confidence interval), 1.15-5.20) after multivariate adjustment for confounding clinical factors. On the analyses of low-risk PTMC, recurrences were 1.3% (5/383) versus 4.3% (6/139) in BRAF mutation-negative versus BRAF mutation-positive patients, with an HR of 6.65 (95% CI, 1.80-24.65) after adjustment for confounding clinical factors. BRAF mutation was associated with a significant decline in the Kaplan-Meier recurrence-free survival curve in low-risk PTMC., Conclusions: BRAF V600E differentiates the recurrence risk of PTMC, particularly low-risk PTMC. Given the robust negative predictive value, conservative active surveillance of BRAF mutation-negative low-risk PTMC is reasonable whereas the increased recurrence risk and other well-known adverse effects of BRAF V600E make the feasibility of long-term conservative surveillance uncertain for BRAF mutation-positive PTMC., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Precise risk stratification-based treatment of solitary intrathyroidal papillary thyroid cancer (SI-PTC) that is larger than 1.0 cm and 4.0 cm or less is undefined., Methods: A genetic-clinical risk study was performed on BRAF V600E in 955 patients (768 women and 187 men) with SI-PTC, with median age of 46 years and median clinical follow-up time of 64 months at 11 medical centers in six countries. The chi-square test or, for analyses with small numbers, Fisher's exact test was performed to compare recurrence rates. Recurrence-free probability was estimated by Kaplan-Meier (KM) analysis, and the independent effect of BRAF mutation on the recurrence was analyzed by Cox regression and Cox proportional hazard analyses. All statistical tests were two-sided., Results: Recurrence of SI-PTC larger than 1.0 cm and 4.0 cm or less was 9.5% (21/221) vs 3.4% (11/319) in BRAF mutation vs wild-type BRAF patients, with a hazard ratio (HR) of 3.03 (95% confidence interval [CI] = 1.46 to 6.30) and a patient age- and sex-adjusted hazard ratio of 3.10 (95% CI = 1.49 to 6.45, P = .002). Recurrence rates of SI-PTC larger than 2.0 cm and 4.0 cm or less were 16.5% (13/79) vs 3.6% (5/139) in mutation vs wild-type patients (HR = 5.44, 95% CI = 1.93 to 15.34; and adjusted HR = 5.58, 95% CI = 1.96 to 15.85, P = .001). Recurrence rates of SI-PTC larger than 3.0 cm and 4 cm or less were 30.0% (6/20) vs 1.9% (1/54) in mutation vs wild-type patients (HR = 18.40, 95% CI = 2.21 to 152.98; and adjusted HR = 14.73, 95% CI = 1.74 to 124.80, P = .01). Recurrences of mutation-positive SI-PTC were comparable with those of counterpart invasive solitary PTC, around 20% to 30%, in tumors larger than 2.0 cm to 3.0 cm. BRAF mutation was associated with a statistically significant decrease in recurrence-free patient survival on KM analysis, particularly in SI-PTC larger than 2.0 cm and 4.0 cm or less. Similar results were obtained in conventional SI-PTC. The negative predictive values of BRAF mutation for recurrence were 97.8% (95% CI = 96.3% to 98.8%) for general SI-PTC and 98.2% (95% CI = 96.3% to 99.3%) for conventional SI-PTC., Conclusions: BRAF V600E identifies a subgroup of SI-PTC larger than 1.0 cm and 4.0 cm or less, particularly tumors larger than 2.0 cm and 4.0 cm or less, that has high risk for recurrence comparable with that of invasive solitary PTC, making more aggressive treatment reasonable.

Background: Multicellular spheroids represent an interesting experimental model with promising applications in the pre-clinical studies on anticancer drugs. We recently demonstrated that thyroid spheroids recapitulate the features of the original tissues, in either the differentiated and "stem-like" components. Here we were aimed to characterize thyroid spheroids and to investigate in vivo the proangiogenic potential of patient-derived xenografts (PDX) of spheroids obtained from papillary thyroid cancer (PTC) and the matched normal tissues., Methods: Spheroids cultures were obtained from 11 PTCs and matched normal tissues and characterized by immunohistochemistry. The expression of p53, involved in the regulation of stem cell homeostasis, was evaluated. The proangiogenic effect of thyroid spheroids was assessed by the injection in zebrafish embryos., Results: Thyroid spheroids are enriched in stem-like cells, as shown by the positivity for the stem cell marker OCT4, and by the low level of p53 expression. Interestingly, PTCs and normal thyroid tissues have a detectable p53 expression, whereas the derived spheroids are mainly constituted by cells that express p53 at a lower level. Finally, we show that PDXs derived from PTC or normal spheroids stimulate the migration and the growth of sprouting vessels toward the implant into the zebrafish embryos., Conclusions: We report the characterization of multicellular spheroids obtained from PTCs and normal thyroid tissues, showing that they are enriched in stem-like cells. Moreover, we established xenografts of spheroids in zebrafish, demonstrating that they stimulate neoangiogenesis. This in vivo model could be considered as a valuable platform to test the effects of anticancer drugs.

Objectives: The goal of evidence-based practice guidelines is to optimize the management of emerging diseases, such as differentiated thyroid cancer (DTC). The aim of this study was to assess therapeutic approaches for DTC in Italy and to see how closely these practices conformed to those recommended in the 2009 American Thyroid Association (ATA) guidelines., Methods: The Italian Thyroid Cancer Observatory was established to collect data prospectively on thyroid cancers consecutively diagnosed in participating centers (uniformly distributed across the nation). Data on the initial treatment of all pathologically confirmed DTC cases present in the database from January 1, 2013 (database creation) to January 31, 2016, were analyzed., Results: A total of 1748 patients (77.2% females; median age 48.1 years [range 10-85 years]) were enrolled in the study. Most (n = 1640; 93.8%) were papillary carcinomas (including 84 poorly differentiated/aggressive variants); 6.2% (n = 108) were follicular and Hürthle cell carcinomas. The median tumor diameter was 11 mm (range 1-93 mm). Tumors were multifocal in 613 (35%) and presented extrathyroidal extension in 492 (28%) cases. Initial treatments included total thyroidectomy (involving one or two procedures; n = 726; 98.8%) and lobectomy (n = 22; 1.2%). A quarter of the patients who underwent total thyroidectomy had unifocal, intrathyroidal tumors ≤1 cm (n = 408; 23.6%). Neck dissection was performed in 40.4% of the patients (29.5% had central compartment dissection). Radioiodine remnant ablation (RRA) was performed in 1057 (61.2%) of the 1726 patients who underwent total thyroidectomy: 460 (41.2%) of the 983 classified by 2009 ATA guideline criteria as low-risk, 570 (87.1%) of the 655 as intermediate-risk, and 82 (93.1%) of the 88 as high-risk patients (p < 0.001). RRA was performed in 44% of the cases involving multifocal DTCs measuring ≤1 cm., Conclusions: The treatment approaches for DTCs used in Italy display areas of inconsistency with those recommended by the 2009 ATA guidelines. Italian practices were characterized by underuse of thyroid lobectomy in intrathyroidal, unifocal DTCs ≤1 cm. The use of RRA was generally consistent with risk-stratified recommendations. However, its frequent use in small DTCs (≤1 cm) that are multifocal persists, despite the lack of evidence of benefit. These data provide a baseline for future assessments of the impact of international guidelines on DTC management in Italy. These findings also illustrate that the dissemination and implementation of guideline recommendations, and the change in practice patterns, require ongoing education and time.

Context: Multifocality is often treated as a risk factor for papillary thyroid cancer (PTC), prompting aggressive treatments, but its prognostic value remains unestablished., Objective: To investigate the role of tumor multifocality in clinical outcomes of PTC., Methods: Multicenter study of the relationship between multifocality and clinical outcomes of PTC in 2638 patients (623 men and 2015 women) with median [interquartile range (IQR)] age of 46 (35 to 58) years and median (IQR) follow-up time of 58 (26 to 107) months at 11 medical centers in six countries. Surveillance, Epidemiology and End Results (SEER) data were used for validation., Results: Disease recurrence in multifocal and unifocal PTC was 198 of 1000 (19.8%) and 221 of 1624 (13.6%) (P < 0.001), with a hazard ratio of 1.55 [95% confidence interval (CI), 1.28 to 1.88], which became insignificant at 1.13 (95% CI, 0.93 to 1.37) on multivariate adjustment. Similar results were obtained in PTC variants: conventional PTC, follicular-variant PTC, tall-cell PTC, and papillary thyroid microcarcinoma. There was no association between multifocality and mortality in any of these PTC settings, whereas there was a strong association between classic risk factors and cancer recurrence or mortality, which remained significant after multivariate adjustment. In 1423 patients with intrathyroidal PTC, disease recurrence was 20 of 455 (4.4%) and 41 of 967 (4.2%) (P = 0.892) and mortality was 0 of 455 (0.0%) and 3 of 967 (0.3%) (P = 0.556) in multifocal and unifocal PTC, respectively. The results were reproduced in 89,680 patients with PTC in the SEER database., Conclusions: Tumor multifocality has no independent risk prognostic value in clinical outcomes of PTC; its indiscriminate use as an independent risk factor, prompting overtreatments of patients, should be avoided., (Copyright © 2017 Endocrine Society)

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients' tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and "stem-like" components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and β-catenin turnover. Consistently, SP treatment resulted in a significant decrease in β-catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells.In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.

During hormonogenesis, thyrocytes are physiologically exposed to high levels of oxidative stress (OS) which could either be involved in the pathogenesis of thyroid cancer or exert a cytotoxic effect. We analyzed the oxidative status of papillary thyroid cancer (PTC) both directly, by measuring H2O2 generation by NADPH oxidases (NOXs), and indirectly, by evaluating the antioxidant activity of glutathione peroxidase (GPX), which neutralizes H2O2 excess, and the lipid peroxidation (LP). Moreover, we investigated the subcellular localization of telomerase reverse transcriptase (TERT), and the H2O2 levels in the mitochondria of tumor and normal tissues. The calcium-dependent and independent H2O2 generation activity was significantly higher in tumors than in normal tissues. The GPX activity was higher in PTCs than in normal tissues, and, consistently, no differences were found in LP levels. Moreover, while TERT nuclear expression was similar in tumor and normal tissues, the mitochondrial localization was significantly higher in tumors. At the mitochondrial level, no differences were found in H2O2 generation between tumor and normal tissues. In conclusion, present data demonstrate that the intracellular H2O2 generation by NOXs is significantly higher in PTCs than in normal thyroid tissues. The increased GPX activity found in tumors counteracts the potential cytotoxic effects of high OS exposure. The significantly higher mitochondrial localization of TERT in tumors is consistent with its shuttling from the nucleus upon exposure to high OS. Finally, mitochondrial OS was not significantly different in tumors and normal tissues, supporting the postulated role of mitochondrial TERT in the control of local H2O2 production., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Fetal cell microchimerism (FCM) is defined as the persistence of fetal cells in maternal organs and circulation without any apparent rejection and it was hypothesized to protect toward the onset of some neoplastic diseases. To verify the role of FCM in papillary thyroid cancer (PTC), we enrolled 87 parous women with PTC and at least one male pregnancy preceding the diagnosis (PTC-P), 66 healthy women with 1 or more male children (HC-P) and 57 nonparous women with PTC (PTC-NP). 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/1 million female cells. A significantly higher frequency of FCM was found in HC-P than PTC-P women (63.6% vs. 39.1%, p = 0.004). Among PTC-P patients, those positive for the presence of FCM (FMC+ve) had a lower prevalence of extrathyroidal extension (p = 0.027) and lymph node metastases (p = 0.044) than those without FCM (FMC-ve). Moreover, FMC+ve patients were more frequently in remission than FMC-ve cases (94.1 vs. 67.9%, p = 0.009). Interestingly, we showed for the first time that the positive effect on tumor presentation and outcome is specifically related to FCM and it is not an effect of pregnancy. In conclusion, circulating FCM is significantly more frequent in healthy parous women than in women with PTC. Moreover, the presence of circulating fetal male cells is associated with a significantly lower extrathyroidal extension and a good prognosis, suggesting a protective role of this phenomenon toward both the onset and the progression of thyroid cancer., (© 2015 UICC.)

Effective adverse event (AE) management is critical to maintaining patients on anticancer therapies. The DECISION trial was a multicenter, randomized, double-blind, placebo-controlled, Phase 3 trial which investigated sorafenib for treatment of progressive, advanced, or metastatic radioactive iodine-refractory, differentiated thyroid carcinoma. Four hundred and seventeen adult patients were randomized (1:1) to receive oral sorafenib (400 mg, twice daily) or placebo, until progression, unacceptable toxicity, noncompliance, or withdrawal. Progression-free survival, the primary endpoint of DECISION, was reported previously. To elucidate the patterns and management of AEs in sorafenib-treated patients in the DECISION trial, this report describes detailed, by-treatment-cycle analyses of the incidence, prevalence, and severity of hand-foot skin reaction (HFSR), rash/desquamation, hypertension, diarrhea, fatigue, weight loss, increased serum thyroid stimulating hormone, and hypocalcemia, as well as the interventions used to manage these AEs. By-cycle incidence of the above-selected AEs with sorafenib was generally highest in cycle 1 or 2 then decreased. AE prevalence generally increased over cycles 2-6 then stabilized or declined. Among these AEs, only weight loss tended to increase in severity (from grade 1 to 2) over time; severity of HFSR and rash/desquamation declined over time. AEs were mostly grade 1 or 2, and were generally managed with dose interruptions/reductions, and concomitant medications (e.g. antidiarrheals, antihypertensives, dermatologic preparations). Most dose interruptions/reductions occurred in early cycles. In conclusion, AEs with sorafenib in DECISION were typically grade 1 or 2, occurred early during the treatment course, and were manageable over time., (© 2015 The authors.)

Background: Thyroid cancer is highly prevalent in women during the fertile age, which suggests a possible impact of hormonal and reproductive factors., Methods: We studied the expression of estrogen receptor α (ERα or ESR1) and progesterone receptor (PR or PGR) in 182 female and male patients with papillary thyroid cancer and correlated it to clinical and molecular features., Results: ERα and PR expression was found in 66.5 and 75.8% of patients respectively and was significantly correlated with larger tumor size and with a non-incidental diagnosis. Moreover, a trend toward a higher prevalence of local metastases was observed in ER- and PR-expressing tumors, which possibly indicates a more aggressive behavior. Interestingly, the occurrence of the 'receptor conversion' phenomenon, which has already been reported to have a negative prognostic effect in breast cancer, was demonstrated for the first time in thyroid tumors. Indeed, almost all of the ERα-positive primary tumors analyzed had ERα-negative metastatic lymph nodes. At the genetic analyses, BRAF(V600E) mutation was detected in 23.2% of the tumors and had a higher prevalence in larger tumors and in those with a stronger ERα or PR staining., Conclusions: The whole of the findings reported in the present study argue for an association between ERα and PR sex hormone receptor expression and a more aggressive presentation. Although no impact on outcome was found, the evaluation of ERα and PR receptor expression could add insights into the biological behavior of tumors and could modify the follow-up, particularly in fertile women affected with persistent disease., (© 2015 European Society of Endocrinology.)

Purpose: To investigate the prognostic value of BRAF V600E mutation for the recurrence of papillary thyroid cancer (PTC)., Patients and Methods: This was a retrospective multicenter study of the relationship between BRAF V600E mutation and recurrence of PTC in 2,099 patients (1,615 women and 484 men), with a median age of 45 years (interquartile range [IQR], 34 to 58 years) and a median follow-up time of 36 months (IQR, 14 to 75 months)., Results: The overall BRAF V600E mutation prevalence was 48.5% (1,017 of 2,099). PTC recurrence occurred in 20.9% (213 of 1,017) of BRAF V600E mutation-positive and 11.6% (125 of 1,082) of BRAF V600E mutation-negative patients. Recurrence rates were 47.71 (95% CI, 41.72 to 54.57) versus 26.03 (95% CI, 21.85 to 31.02) per 1,000 person-years in BRAF mutation-positive versus -negative patients (P < .001), with a hazard ratio (HR) of 1.82 (95% CI, 1.46 to 2.28), which remained significant in a multivariable model adjusting for patient sex and age at diagnosis, medical center, and various conventional pathologic factors. Significant association between BRAF mutation and PTC recurrence was also found in patients with conventionally low-risk disease stage I or II and micro-PTC and within various subtypes of PTC. For example, in BRAF mutation-positive versus -negative follicular-variant PTC, recurrence occurred in 21.3% (19 of 89) and 7.0% (24 of 342) of patients, respectively, with recurrence rates of 53.84 (95% CI, 34.34 to 84.40) versus 19.47 (95% CI, 13.05 to 29.04) per 1,000 person-years (P < .001) and an HR of 3.20 (95% CI, 1.46 to 7.02) after adjustment for clinicopathologic factors. BRAF mutation was associated with poorer recurrence-free probability in Kaplan-Meier survival analyses in various clinicopathologic categories., Conclusion: This large multicenter study demonstrates an independent prognostic value of BRAF V600E mutation for PTC recurrence in various clinicopathologic categories., (© 2014 by American Society of Clinical Oncology.)

Unlabelled: Post-operative neck nodal metastases are detected in 9-20 % of papillary thyroid cancer (PTC). Ultrasound-guided ethanol injection (UPEI) has been used with promising results in the treatment of PTC patients with limited number of lymph-node metastases., Clinical Case: We report the imaging and biochemical documentation of UPEI results in four metastatic neck lymph-nodes of papillary thyroid-cancer. A significant reduction both in the diameter of the lymph-nodes and in the serum thyroglobulin levels were observed in all cases and have been reported in a step-by-step sequence., Conclusion: the UPEI treatment is a simple and suitable tool for the treatment of PTC cervical lymph-node metastases.

Purpose: The aim of this study was to evaluate the efficacy of post-operative radioiodine ablation with 1,850 MBq after recombinant human thyrotropin (rhTSH) administration in patients with differentiated thyroid carcinoma (DTC). We also aimed to assess the prognostic role of several patient features on the outcome of ablation., Methods: We retrospectively analyzed data from a total of 125 patients with DTC who underwent post-operative radioiodine ablation with 1,850 MBq of ¹³¹I after preparation with rhTSH. One injection of 0.9 mg rhTSH was administered on each of two consecutive days; ¹³¹I therapy was delivered 24 h after the last injection, followed by a post-therapy whole-body scan. Successful ablation was assessed 6-12 months later and defined as an rhTSH-stimulated serum thyroglobulin (Tg) level ≤1.0 ng/ml and a normal neck ultrasound., Results: Patients were stratified according to the American Thyroid Association (ATA) Management Guidelines for Differentiated Thyroid Cancer. Successful ablation was achieved in 82.4 % of patients, with an ablation rate of 95.1 % in low-risk patients and 76.2 % in intermediate-risk patients. Analyzing the correlation between ablation outcome and patient characteristics, we found a statistically significant association between failure to ablate and class of risk based on ATA guidelines (p = 0.025) and a stimulated Tg value at ablation of above 5 ng/ml (p < 0.001)., Conclusion: The use of 1,850 MBq post-operative radioiodine thyroid remnant ablation in association with rhTSH is effective for low- and intermediate-risk patients. Moreover, in our study, we found a statistical correlation between failure to ablate and class of risk based on ATA guidelines for DTC and a stimulated Tg value at ablation.

Background: Evidence for an increased prevalence of BRAF(V600E) mutations has been documented in recent decades. The aim of this study was to evaluate the prevalence of both RET/PTC rearrangements and BRAF(V600E) mutations in an Italian cohort of papillary thyroid carcinoma (PTC) patients followed at the Endocrine Units of Pisa, Milano, and Perugia from 1996-2010., Patients and Methods: In total, 401 PTC patients were examined and grouped according to the time of surgery: group 1, 1996-2000; group 2, 2001-2005; and group 3, 2006-2010. Patients were analyzed for clinical, pathological, and molecular features. In parallel, the molecular characteristics of 459 PTC from Sicily were studied., Results: The genetic profiles of the three groups were significantly different (P < 0.0001). In particular, the frequency of RET/PTC rearrangements decreased from 1996-2010, occurring in 33 of 100 (33%) of the patients in group 1, 26 of 148 (17%) in group 2, and 15 of 153 (9.8%) in group 3. The incidence of BRAF(V600E) mutations increased over the same period, from 28% in group 1 (28 of 100) to 48.9% in group 2 (73 of 148) and 58.1% in group 3 (89 of 153). A consistent increase in BRAF(V600E) prevalence was observed in the Sicilian group (P < 0.0001). Moreover, a statistically significant increase in the mean age at diagnosis and decrease in tumor size over the study period was observed., Conclusion: The genetic profile of PTC changed over the last 15 yr, with a significant decrease in the prevalence of RET/PTC rearrangements and an increase in BRAF(V600E) mutations. In addition, the mean age at diagnosis increased and tumor size decreased over the study period.

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.

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., Patients: The clinical and molecular features, and the expression of inflammation-related genes, were investigated in a large series of PTCs with and without associated thyroiditis (groups A, n = 128 and B, n = 215)., Results: The two groups did not show significant differences in clinical and prognostic features, whereas they harboured a significantly different genetic background (P = 0.001), with RET/PTC1 being more represented in PTCs associated with autoimmunity, and BRAF(V600E) in patients with PTC alone. A RET/PTC rearrangement was also found in 41% of non-neoplastic thyroiditis tissues, contralateral to tumours harbouring either RET/PTC or BRAF mutations. The expression of genes encoding CCL20, CXCL8 and l-selectin was significantly higher in PTC specimens (either with RET/PTC, BRAF(V600E) or unknown genetic lesion) compared with normal thyroid samples. On the contrary, thyroiditis showed l-selectin expression levels even higher than PTCs, but CCL20 and CXCL8 levels comparable with normal tissues., Conclusions: The present data extend the knowledge about the tight relationships among oncogenes, thyroiditis and thyroid cancer. A different genetic background among PTCs with and without associated autoimmunity has been firstly demonstrated. The strong association between RET/PTC1 and thyroiditis points to a critical role of this oncoprotein in the modulation of the autoimmune response. Moreover, preliminary expression studies, indicating enhanced expression of inflammatory molecules in PTCs, suggest a proinflammatory, nonautoimmune relationship between thyroiditis and thyroid cancer.

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 cells enter the maternal circulation during pregnancy and can persist in the maternal blood or tissues for decades, creating a physiologic microchimerism. Because papillary thyroid cancer (PTC) is more frequent in women, the role of persisting fetal male cells in this tumor has been investigated. Tumor tissue specimens were obtained from 63 women with PTC who had a male pregnancy before the diagnosis. Male cells, identified by PCR amplification of a male-specific gene, the sex-determining region Y, was detected in 47.5% of women. By fluorescence in situ hybridization (FISH) analyses, the total number of microchimeric cells was significantly higher in neoplastic tissue than in controlateral normal sections. By combined FISH and immunohistochemistry (immuno-FISH), male cells expressing thyroglobulin were found in tumor and normal tissues, whereas male microchimeric cells stained with the CD45 antigen were detected only in tumor sections. Microchimeric cells negative for either marker were detected both in tumor and normal tissues. Moreover, both CD45(+) and Tg(+) fetal cells did not express MHC II antigens. In conclusion, fetal microchimerism has been documented in a high proportion of women with PTC. The immuno-FISH studies indicate that CD45(+)/MHC II(-) male cells found in neoplastic tissues might be committed to destroy tumor cells, whereas Tg(+)/MHC II(-) cells could have a repair function. Finally, microchimeric cells negative for either CD45 or Tg could have "progenitor-like" properties able to transdifferentiate in different cellular types. Although a pathogenetic mechanism cannot be excluded, the whole of the present results indicates a protective role of microchimerism in thyroid cancer.

Recently, a somatic point mutation of the B-RAF gene (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence variable among different series. Since discordant data on the clinico-pathologic features of B-RAF mutated PTC are present in the literature, the aim of the present co-operative study was to establish the prevalence of this genetic alteration and to perform a genotype-phenotype correlation in a large cohort of patients with PTC. To this purpose, a series of 260 sporadic PTCs with different histological variants were included in the study. The mutational analysis of the B-RAF gene was performed either by RT-PCR followed by single-stranded conformational polymorphism or by PCR and direct sequencing. Statistical analyses were obtained by means of chi2/Fisher's exact test and t-test. Overall, a heterozygous T > A transversion at nucleotide 1799 (V600E) was found in 99 out of 260 PTCs (38%). According to the histological type of the tumor, the B-RAF (V600E) mutation was present in 48.3% of cases of classic PTCs (85 out of 176), in 17.6% (nine out of 51) of follicular variants of PTCs, in 21.7% (five out of 23) in other PTC variants and in none of the ten poorly differentiated tumors. B-RAF (V600E) was significantly associated with the classic variant of PTC (P = 0.0001) and with an older age at diagnosis (P = 0.01). No statistically significant correlation was found among the presence of B-RAF (V600E) and gender, tumor node metastasis (TNM), multicentricity of the tumor, stage at diagnosis and outcome. In conclusion, the present study reports the prevalence of B-RAF (V600E) (38%) in the largest series of sporadic PTCs, including 260 cases from three different Italian referring centers. This prevalence is similar to that calculated by pooling together all data previously reported, 39.6% (759 out of 1914 cases), thus indicating that the prevalence of this genetic event lies around 38-40%. Furthermore, B-RAF (V600E) was confirmed to be associated with the papillary growth pattern, but not with poorer differentiated PTC variants. A significant association of B-RAF mutation was also found with an older age at diagnosis, the mutation being very rare in childhood and adolescent PTCs. Finally, no correlation was found with a poorer prognosis and a worse outcome after a median follow-up of 72 months.

We report the simultaneous occurrence of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC), presenting as spatially distinct and well-defined tumour components, in three cases. In the first patient, histology, immunohistochemistry and electron microscopy demonstrated an MTC in the one nodule and PTC in two additional lesions. Non-neoplastic thyroid parenchyma separated the three nodules. Metastasis from PTC was diagnosed in a regional lymph node. Genetic analysis of both tumour components showed a distinctive mutational pattern: in the MTC a Cys634Arg substitution in exon 11 of the RET gene and in the two PTC foci a Val600Glu substitution in exon 15 of the BRAF gene. The other two patients are members of a large multigenerational family affected with familial MTC due to a germline mutation of the RET gene (Ala891Ser). Both patients harboured, besides medullary cancer and C-cell hyperplasia, distinct foci of papillary thyroid cancer, which was positive for Val600Glu BRAF mutation. Review of the literature disclosed 18 similar lesions reported and allowed the identification of different patterns of clinical presentation and biological behaviour. So far, the pathogenesis of these peculiar cases of thyroid malignancy has been completely unknown, but an underlying common genetic drive has been hypothesised. This is the first report in which two mutations, in the RET and BRAF genes, have been identified in three cases of MTC/PTC collision tumour, thus documenting the different genetic origin of these two coexisting carcinomas.

Objective: Recently, a somatic point mutation of the BRAF gene (V599E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC) with a variable frequency (about 25-70%) in different series from USA, Japan, Portugal and Ukraine., Design: In the present study, the genetic analysis of BRAF in an Italian cohort of 65 thyroid tumours with corresponding normal tissues and 21 thyroid benign disorders is reported., Methods: For BRAF analysis, the somatic DNA was PCR amplified by means of specific intronic primers and PCR products were directly sequenced. Statistical analyses were obtained by means of Fisher's exact test., Results: All mutations detected involved a T > A transversion at 1796 (V599E) and were heterozygous. Overall, BRAF(V599E) mutation was found in 18/56 (32.1%) PTCs. According to the histological type of the tumour, the mutation was present in 38.3% of cases of conventional PTC (18/47), in 0/6 follicular variant of PTC, in 0/3 oncocytic variant of PTC. No BRAF mutations were detected either in five follicular carcinomas, or in four poorly differentiated or undifferentiated cancers or in benign thyroid disorders. No statistically significant correlation of BRAF mutation with patient age and gender, with multicentricity of the tumour, with the lymphocytic infiltration of the tissue, with the stage and with the recurrence rate, was found. BRAF(V599E) tended to be associated, although not significantly, with a greater volume and extension of the tumour and with lymph-nodal metastases at surgery., Conclusions: In conclusion, the present study on the first Italian series of thyroid cancers shows a frequency of 38.3% of BRAF(V599E) in the classical variant of PTC, confirming the key role of this mutation in promoting tumourigenesis.

Background: Occurrence in a familial setting is well established for medullary thyroid carcinoma (MTC) and has been more recently reported for papillary thyroid cancer (PTC). Germline mutations or rearrangements of the RET proto-oncogene are the genetic background of the majority of hereditary MTCs and of about 25-40% of PTCs., Patients: A large multigenerational familial medullary thyroid cancer (FMTC) family, comprised of four generations and a total of 60 subjects, has been fully evaluated. Studies on germline RET mutations and polymorphisms, on somatic RET activation and on haplotyping with RET-linked markers, were performed., Results: RET mutational analysis revealed a rare missense point mutation in exon 15 of RET (A891S), associated with FMTC. Haplotype analysis showed a co-segregation between the allelic variant 5 of D10S578 marker (which is tightly linked to the RET locus) and the RET mutation. Two patients, from different branches of the family, did not harbour the point mutation A891S despite histological confirmation of MTC. In these cases, haplotype analysis excluded the involvement of the RET gene itself in the pathogenesis of the MTC. In three patients, the coexistence, in different foci, of medullary and papillary thyroid cancer was documented. The genetic studies did not show ret/PTC rearrangements. The microsatellite analysis excluded co-segregation of RET locus with the MTC/PTC phenotype., Conclusions: We report a full clinical and molecular analysis of a large FMTC kindred with an uncommon RET mutation. In two family members, phenotype and genotype were not concordant, representing the first evidence of FMTC phenocopies. Furthermore, the association of familial forms of medullary and papillary thyroid cancers has been found in 30% of patients undergoing thyroidectomy for MTC. In these situations, genetic analyses excluded the possible germline involvement of RET. Though FMTC phenocopies are likely to represent an exceptional finding, such a possibility should be taken into account in the genetic counselling for MEN 2 syndromes.

The RET/PTC3 oncogene is an activated form of the RET protooncogene, which is frequently rearranged in papillary thyroid carcinoma. RET/PTC3 results from a structural rearrangement between the ELE1 and the RET genes, and it has been observed in both sporadic and radiation-associated post-Chernobyl tumors. To understand the molecular basis that predisposes RET and ELE1 genes to be recurrent targets of "illegitimate" recombination, we examined the genomic regions containing the ELE1/RET breakpoints of six sporadic and three post-Chernobyl tumors in two papillary carcinomas of different origins. Our data indicated, in both genes, a clustering of the breakpoints in regions designated ELE1-bcr (1.8 kb) and RET-bcr (1.9 kb). Notably, in all sporadic tumors and in one post-Chernobyl tumor the ELE1/RET recombination corresponded with short sequences of homology (3-7 nt) between the two rearranging genes. In addition, we observed an interesting distribution of the post-Chernobyl breakpoints in ELE1-bcr located within an Alu element, or in between two close Alu elements, and always in A+T-rich regions.

A post-Chernobyl papillary thyroid cancer, displaying a novel ELE1/RET oncogenic rearrangement with an anomalous fusion transcript, was molecularly characterized. In spite of the presence of a normal breakpoint in exon 5 of the activating ELE1 gene, the sequence of the rearranged genomic DNA showed a previously unreported intra-exonic breakpoint in the RET protooncogene. As a consequence, a cDNA sequence 93 nucleotides larger than the regular one, and with the exon 5 of ELE1 joined to exon 11 instead of exon 12 of RET, is formed. To characterize the product of this new oncogenic ELE1/RET rearrangement, here designated as RET/PTC4, we performed an immunoprecipitation and Western blot analysis on cell extracts from NIH3T3 transfectants. The results showed the presence of two isoforms of the chimeric protein, displaying a constitutive tyrosine phosphorylation. As expected, the molecular weight of this protein was higher than that of RET/ PTC3 protein (p80 and p85, instead of p76 and p81). Previous reports, from our and other laboratories, showed that post-Chernobyl papillary thyroid carcinomas are characterized by a high frequency (about 60%) of RET oncogenic rearrangements (Fugazzola et al., 1995; Klugbauer et al., 1995; Ito et al., 1994). These events predominantly involve ELE1 activating sequence, thus producing RET/PTC3 oncogene (Fugazzola et al., 1995; Klugbauer et al., 1995). Hence, this elevated frequency of RET rearrangements could increase the probability of selecting unusual events as that here described. Alternatively, targeted radiation effects could be responsible for the atypical RET rearrangement producing RET/PTC4 oncogene.

Oncogenic rearrangements of RET and NTRK1 proto-oncogenes are frequently detected in papillary thyroid carcinomas. Several studies have shown an association between ionizing radiation and development of this tumor type. In addition in vitro irradiation of tumor cell lines induced rearrangements of RET similar to those observed in human papillary thyroid carcinomas. These two observations could be related to the reported increased incidence of papillary thyroid carcinomas in children living in contaminated areas around Chernobyl, given that it has been demonstrated that about 60% of them presents a RET oncogenic activation. However, this high frequency of RET positivity in radiation exposed children does not rule out the possibility that age could also play a role in the development of RET positive tumors. To assess this possibility we looked for a relationship between the presence of RET and NTRK1 oncogenic rearrangements and age at surgery in a sample of 92 consecutive patients. Our results show that, in papillary thyroid carcinoma, the frequency of RET and NTRK1 activation is significantly higher in the group of patients aged 4-30 years, thas supporting the concept that age could be contributing to this thyroid specific carcinogenic process.

Since the Chernobyl nuclear reactor accident, a striking increase of thyroid carcinoma has been reported in children exposed to radiation in Belarus. Because of its unprecedented scale and its emotional implications, this finding has raised concern and called the attention of the scientific community to this major health problem. Although epidemiologically documented, a direct correlation between thyroid cancer and radiation exposure has not been definitely proven at the molecular level. On the assumption that ionizing radiation could cause specific and common cancer-associated genetic lesions, an analysis of oncogene activation and/or tumor suppressor gene inactivation would help to define radiation-induced thyroid carcinomas. Therefore, we have analyzed by different molecular approaches, including Southern blotting, DNA transfection assay on NIH-3T3 cells, and reverse transcription-PCR analysis, six papillary carcinomas from children living in the region of Belarus at the time of the Chernobyl nuclear accident to identify tumor-specific gene rearrangements of the proto-oncogenes RET and TRK, previously found activated in a tumor type-specific manner in papillary thyroid carcinoma. Using Southern blot analysis in four cases, we could detect specific rearranged bands indicating an oncogenic activation of RET that in three cases resulted in rearranged sequences provided by the same activating gene. Moreover, the DNA of the last three cases showed a biological activity in transforming NIH-3T3 cells after the DNA-mediated transfection assay, and the respective NIH-3T3 transfectants were found to express the oncogenic fusion transcripts. These results support the possibility that RET oncogenic activation could represent a major genetic lesion associated with thyroid carcinoma in children exposed to the Chernobyl nuclear accident.

Increased concentrations of serum soluble interleukin-2 (IL-2) receptor (sIL-2R), a marker of T-lymphocyte activation, have been found in several metastatic solid tumors. To our knowledge, no information is available on serum sIL-2R in differentiated thyroid carcinoma (DTC). Aim of this study was to evaluate whether disease activity and/or thyroid status may affect circulating sIL-2R in DTC, since it is has recently been demonstrated that serum thyroid hormone concentration positively modulates circulating sIL-2R. DTC patients were divided into 3 groups: Group A: 48 patients without metastases or local recurrences; Group B: 16 patients with cervical lymph node metastases; Group C: 22 patients with distant metastases. All patients were evaluated after total thyroidectomy both off and on L-thyroxine (L-T4) therapy. Control group was composed by 20 healthy euthyroid subjects. sIL-2R was assayed by solid-phase ELISA. In the hypothyroid state, sIL-2R levels of Group A were significantly lower when compared to normal controls (256 +/- 130 vs. 461 +/- 186 U/ml, p < 0.001 by Student t test); Group B and Group C patients off L-T4 therapy had sIL-2R concentrations significantly higher (479 +/- 407 U/ml, Group B; 519 +/- 746 U/ml, Group C) when compared to hypothyroid patients of Group A (p < 0.01 and p < 0.05, respectively), but not significantly different from normal euthyroid controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlabelled: The aim of the present study was to assess whether 131I therapy for differentiated thyroid carcinoma (DTC) can affect endocrine testicular function., Methods: Serum follicle-stimulating hormone (FSH) and testosterone (T) concentrations were measured in 103 patients periodically submitted for radioiodine therapy for residual or metastatic disease. Mean follow-up was 93.7 +/- 54 mo (range 10-243 mo)., Results: Mean FSH values in 131I-treated patients tested after their last treatment were 15.3 +/- 9.9 mU/ml, significantly higher than those of 19 untreated patients (6.5 +/- 3.1 mU/ml). Considering the mean +3 s.d. FSH of untreated subjects as the upper limit of normal range, 36.8% of the patients had an abnormal increase in serum FSH. Longitudinal analysis performed in 21 patients showed that the behavior of FSH in response to 131I therapy was not universal. Six patients had no change or a slight increase in serum FSH after 131I administration; eleven patients had a transient increase above normal values 6-12 mo after 131I treatment, with return to normal levels in subsequent months. The administration of a second dose was followed by a similar increase in FSH levels. Finally, four patients, followed for a long period of time and treated with several 131I doses, showed a progressive increase in serum FSH, which eventually became permanent. Semen analysis, performed in a small subgroup of patients, showed a consistent reduction in the number of normokinetic sperm. No change was found in serum T levels between treated and untreated patients., Conclusions: Our results indicate that 131I therapy for thyroid carcinoma is associated with transient impairment of testicular germinal cell function. The damage may become permanent for high-radiation activities delivered year after year and might pose a significant risk of infertility.

We studied the expression of p21, the ras encoded protein, in primary tumour of 45 patients with papillary thyroid cancer (PTC). Patients were grouped according to outcome so that one group (31 patients) had a good outcome and the other (14 patients) a fatal outcome, after a follow-up of at least 5 years. The presence of p21 ras protein was assessed by immunohistochemistry with a specific monoclonal antibody (MAb Y-13259). The results were correlated with the outcome, with the expression of proliferating cell nuclear antigen (PCNA)/cyclin (as a marker of cell proliferation) and with other well established prognostic factors for PTC (age, grading, extension and tumour size; Endocrinol Metab Clin North Am 1990, 19, 545-576). p21 staining in tumours of living patients was negative in 15, weakly positive (1+) in 10 and strongly positive (2+ or more) in 6 patients. In tumours from deceased patients, p21 staining was negative in 1, weakly positive in 2 and strongly positive in the remaining 11 patients (P < 0.001, chi 2). PCNA immunostaining was increased in 63.6% (7/11) of the tumours from deceased patients compared to 17.8% (5/28) of the tumours of living patients, but no direct correlation was found between p21 and PCNA expression. Among the other prognostic factors studied, only age > or = 40 years was a significant predictor of poor outcome. The survival curve of patients with strongly positive p21 staining was similar to that of patients aged > or = 40 years at the time of diagnosis. The combination of p21 > or = 2+ and age > or = 40 was superior to age alone (P < 0.05) as a prognostic indicator of poor outcome. In conclusion, our results indicate that the p21 product of the ras (proto)oncogene is differently expressed in PTC, in relation to the degree of aggressiveness. Regardless of the pathogenetic role of the ras oncogene in thyroid tumorigenesis, our data indicate that the expression of the p21 ras protein may be regarded as a prognostic indicator in PTC. Furthermore, overexpression of p21 ras protein is associated with patients in the older age groups, and might contribute to the poor prognosis of elderly patients.