Marco Capezzone

Impact of Proto-Oncogene Mutation Detection in Cytological Specimens from Thyroid Nodules Improves the Diagnostic Accuracy of Cytology

CONTEXTnFine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions.nnnOBJECTIVEnWe aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC.nnnSTUDY DESIGNnOne hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing.nnnRESULTSnMutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%).nnnCONCLUSIONSnOur findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.

Familial Non-Medullary Thyroid Carcinoma Displays the Features of Clinical Anticipation Suggestive of a Distinct Biological Entity.

Non-medullary thyroid carcinoma (NMTC) is mostly sporadic, but familial clustering is described. We aimed to compare the features of patients with sporadic and familial NMTC (FNMTC) patients and to assess whether FNMTC patients with parent-child relationship exhibit the anticipation phenomenon (earlier age at disease onset and increased severity in successive generations). Among 300 NMTCs followed in the Section of Endocrinology (University of Siena, Italy), 34 (11.3%) patients, all with the papillary histotype, (16 kindred), met the criteria of FNMTC. Twenty-seven of them (79.4%) exhibited a parent-child relationship and seven (20.6%) a sibling relationship. These patients were compared with 235 patients with sporadic papillary thyroid cancer (PTCs). To analyze the features of FNMTC of the first and second generations, we cumulated the series of Siena with 32 additional FNMTC patients (15 kindred) from the Department of Endocrinology-Endocrine Oncology, Thessaloniki, Greece. Significant difference between sporadic PTC and FNMTC patients included more frequent tumor multifocality (P=0.001) and worse final outcome in FNMTC patients (P=0.001). Among 47 FNMTC with parent-child relationship, we found an earlier age at disease presentation (P<0.0001), diagnosis (P<0.0001), and disease onset (P=0.04) in the second generation when compared with the first generation. Patients in the second generation were more frequently males (P=0.02); their tumors were more frequently multifocal (P=0.003) and bilateral (P=0.01), had higher rate of lymph node metastases at surgery (P=0.02) and worse outcome (P=0.04) when compared with the first generation. In conclusion, FNMTC displays the features of clinical anticipation with the second generation acquiring the disease at an earlier age and having more advanced disease at presentation.

Short telomeres, telomerase reverse transcriptase gene amplification, and increased telomerase activity in the blood of familial papillary thyroid cancer patients

BACKGROUNDnDifferentiated papillary thyroid cancer is mostly sporadic, but the recurrence of the familial form has been reported. Short or dysfunctional telomeres have been associated with familial benign diseases and familial breast cancer.nnnOBJECTIVEnThe aim of our work was to study the telomere-telomerase complex in the peripheral blood of patients with familial papillary thyroid cancer (FPTC), including the measurement of relative telomere length (RTL), telomerase reverse transcriptase (hTERT) gene amplification, hTERT mRNA expression, telomerase protein activity, and search of hTERT or telomerase RNA component gene mutations.nnnPATIENTSnCumulating a series of patients seen at the University of Siena and a series at the University of Rome, the experiments were conducted in 47 FPTC patients, 75 sporadic papillary thyroid cancer (PTC) patients, 20 patients with nodular goiter, 19 healthy subjects, and 20 unaffected siblings of FPTC patients.nnnRESULTSnRTL, measured by quantitative PCR, was significantly (P < 0.0001) shorter in the blood of FPTC patients, compared with sporadic PTCs, healthy subjects, nodular goiter subjects, and unaffected siblings. Also by fluorescence in situ hybridization analysis, the results confirmed shorter telomere lengths in FPTC patients (P = 0.01). hTERT gene amplification was significantly (P < 0.0001) higher in FPTC patients, compared with the other groups, and in particular, it was significantly (P = 0.03) greater in offspring with respect to parents. hTERT mRNA expression, as well as telomerase activity, was significantly higher (P = 0.0003 and P < 0.0001, respectively) in FPTC patients, compared with sporadic PTCs. RTL, measured in cancer tissues, was shorter (P < 0.0001) in FPTC patients, compared with sporadic PTCs. No mutations of the telomerase RNA component and hTERT genes were found.nnnCONCLUSIONnOur study demonstrates that patients with FPTC display an imbalance of the telomere-telomerase complex in the peripheral blood, characterized by short telomeres, hTERT gene amplification, and expression. These features may be implicated in the inherited predisposition to develop FPTC.

Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer

INTRODUCTIONnGenomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex.nnnAIMnWe aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility.nnnMETHODSnTo this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects.nnnRESULTSnWe demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common spontaneous chromosome fragility pattern. A quantitative fluorescence in situ hybridization analysis demonstrates that familial cases display a significant decrease in the telomeric peptide nucleic acid-fluorescence in situ hybridization signal intensity in the metaphase chromosome. Moreover, three copies of the hTERT gene were found only in familial cases, although the result was not statistically significant.nnnCONCLUSIONSnThese results contribute in defining familial thyroid cancer as a clinical entity characterized by an altered telomere stability, which may be associated with the predisposition to develop the familial form of thyroid cancer.

Telomere Length in Neoplastic and Nonneoplastic Tissues of Patients with Familial and Sporadic Papillary Thyroid Cancer

INTRODUCTIONnMany studies have found an association between altered telomere length (TL), both attrition or elongation, and cancer phenotype. Recently, we have reported that patients with the familial form of papillary thyroid cancer (FPTC) have short telomeres in blood leucocytes.nnnAIMnTo evaluate relative TL (RTL) at somatic level in neoplastic and nonneoplastic tissues of patients with FPTC (n = 30) and sporadic PTC (n = 46).nnnMETHODSnRTL was measured by quantitative PCR in neoplastic thyroid tissues, in the corresponding nontumor thyroid tissues (normal contralateral thyroid), and in other extrathyroidal tissues (lymph nodes, muscles, or buccal mucosa). RTL was also measured in adenomas and hyperplastic nodules. In a subset of samples, telomerase expression was measured by quantitative PCR.nnnRESULTSnMean ± SD RTL of FPTC patients was short in neoplastic thyroid tissues (0.87 ± 0.2) with no difference from the normal contralateral thyroid tissues (0.85 ± 0.11) and extrathyroidal tissues (0.85 ± 0.31). On the contrary, in patients with sporadic PTC, the mean ± SD RTL in the neoplastic tissues (1.73 ± 0.63) was significantly shorter than that found in normal contralateral tissues (2.58 ± 0.89) and extrathyroidal tissues (2.5 ± 0.86). For all tissue samples (cancer, normal thyroid, and nonthyroidal tissues) the mean ± SD RTL of familial cases was shorter (P < 0.0001) than that found in tissues from sporadic PTC. RTL of FPTC was also lower (P < 0.0001) than that of 23 follicular adenomas (1.6 ± 0.7) and 24 hyperplastic nodules (2.2 ± 0.9).nnnCONCLUSIONSnOur results demonstrate that short telomeres are a consistent feature of PTC, which in familial cases, is not restricted to the tumor tissue. This finding suggests that FPTC has a distinct, heritable, genetic background.

Lack of germline A339V mutation in thyroid transcription factor-1 (TITF-1/NKX2.1) gene in familial papillary thyroid cancer

Thyroid cancer may have a familial predisposition but a specific germline alteration responsible for the disease has not been discovered yet. We have shown that familial papillary thyroid cancer (FPTC) patients have an imbalance in telomere-telomerase complex with short telomeres and increased telomerase activity. A germline mutation (A339V) in thyroid transcription factor-1 has been described in patients with multinodular goiter and papillary thyroid cancer. In this report, the presence of the A339V mutation and the telomere length has been studied in FPTC patients and unaffected family members. All samples analyzed displayed a pattern typical of the homozygous wild type revealing the absence of the A339V mutation. Shortening of telomeres was confirmed in all patients. We concluded that the A339V mutation in thyroid transcription factor-1 (TITF-1/NKX2.1) is not correlated with the familial form of PTC, even when the tumor was in the context of multinodular goiter.

Telomerase and the endocrine system

Telomeres are nucleoprotein complexes located at the ends of chromosomes that have a critical role in the maintenance of chromosomal integrity. This involvement is based on complex secondary and tertiary structures that rely on DNA–DNA, DNA–protein and protein–protein interactions. De novo synthesis and maintenance of telomere repeats is controlled by telomerase, a specialized complex that consists of a telomerase RNA component and a protein component—telomerase reverse transcriptase. When telomerase is silent (its default state in differentiated somatic cells), chromosomes shorten with every cell division, thus limiting the lifespan of the cells (the process of senescence) and preventing unlimited cell proliferation, which might eventually lead to the development of cancer. During this process, occasionally, a cell can activate telomerase, which stabilizes short telomeres and enables immortalization—a process essential for malignant transformation. Thus, although telomere erosion is a barrier to malignant progression, paradoxically, in certain circumstances it might also trigger tumorigenesis. A number of studies have demonstrated unequivocally that reactivation of telomerase in the presence of short telomeres is one of the most common features of human cancers, including those of the endocrine system.

Small papillary thyroid carcinoma with minimal extrathyroidal extension should be managed as ATA low-risk tumor

PurposeAccording to American Thyroid Association (ATA) guideline, papillary thyroid cancer (PTC) with minimal extrathyroidal extension (mETE) is classified at “intermediate risk” of persistent/recurrent disease. However, the impact of mETE per se on patients’ outcome is not fully understood. The aim of our study was to evaluate the prognostic significance of mETE in patients with PTC not submitted to therapeutic or prophylactic lymph node dissection, according to tumor size and other prognostic factors.Patients and methodsWe retrospectively evaluated a total of 514 PTC patients: 127 (24.7%) had mETE (pT3Nx) and 387 (75.3%) had negative margins (pT1-2Nx). At a median follow-up of 9.1xa0years, patients were divided in two groups: patients with “good outcome” (no evidence of disease) and patients with “poor outcome” (persistent structural disease or recurrent disease or tumor-related death).ResultsThe rate of patients with “poor outcome” was significantly higher in patients with mETE compared with patients with negative margins (11.8 versus 5.1%; OR 2.4576, 95% CI 1.2178–4.9594, pxa0=xa00.01). However, mETE was significantly associated with poor outcome only in patients with tumors larger than 1.5xa0cm.ConclusionsmETE is an unfavorable prognostic factor in tumors larger than 1.5xa0cm, suggesting that, in the absence of other unfavorable characteristics, smaller tumors with mETE should be classified and managed as “low risk” tumors.

Prospective Validation of ATA and ETA Sonographic Pattern Risk of Thyroid Nodules Selected for FNAC

ContextnRecently, the American Thyroid Association (ATA) and the European Thyroid Association (ETA) have proposed that thyroid ultrasound (US) should be used to stratify the risk of malignancy in thyroid nodules and to aid decision-making about whether fine-needle aspiration cytology (FNAC) is indicated.nnnObjectivenTo validate and to compare the ATA and ETA US risk stratification systems of thyroid nodules in a prospective series of thyroid nodules submitted to FNAC.nnnSettingnWe prospectively evaluated 432 thyroid nodules selected for FNAC from 340 patients. Cytology reports were based on the five categories according to the criteria of the British Thyroid Association.nnnResultsnThe proportion of Thy2 nodules decreased significantly, whereas the proportion of Thy4/Thy5 nodules significantly increased with increasing US risk class (P < 0.0001). The ability to identify benign and malignant nodules was similar between ATA and ETA systems. According to ATA and ETA US risk stratification systems, 23.7% and 56.0% nodules did not meet the criteria for FNAC, respectively. Considering only categories at lower risk of malignancy, the cumulative malignancy rate in these nodules was 1.2% for ATA and 1.7% for ETA US risk stratification systems.nnnConclusionsnETA and ATA US risk stratification systems provide effective malignancy risk stratification for thyroid nodules. In clinical practice, using this approach, we should be able to reduce the number of unnecessary FNAC without losing clinically relevant thyroid cancer.

Microcarcinoma papillare della tiroide: il piùfrequente dei tumori endocrini

RiassuntoIl microcarcinoma papillare della tiroide è generalmente un tumore con un’ottima prognosi, ma con un comportamento clinico variabile, tra forme indolenti e prive di rischi nella maggioranza dei casi, a forme clinicamente rilevanti, tuttavia osservate solo in una minoranza di casi. La mancanza di studi prospettici randomizzati rende difficile la possibilità di verificare quale sia l’approccio terapeutico migliore e rende ragione delle incertezze e delle controversie esistenti. Questa rassegna cerca di fare il punto sull’atteggiamento diagnostico-terapeutico più idoneo, attraverso una revisione critica della letteratura.