Prevalence of RET/PTC rearrangement in benign and malignant thyroid nodules and its clinical application.

Fine-needle aspiration cytology (FNAC) is the primary means to distinguish benign thyroid nodules from malignant ones. About 20% of FNAC yields indeterminate results leading to unnecessary or delayed surgery. Many studies of tissue samples, the majority of which are retrospective advocate testing for RET rearrangements as a diagnostic adjunctive tool in thyroid nodules with indeterminate cytological findings. Because of the uncertain prevalence of RET rearrangements, its utility as a tumor marker is still controversial. The goal of this study was to establish the prevalence and the utility of testing for RET rearrangements in FNAC suspicious of cancer in a clinical setting. In this prospective study, we analysed a large series of thyroid aspirates by RT-PCR only and Southern blot on RT-PCR products for type 1 and 3 RET rearrangements. Results were compared with clinical findings, cytological diagnosis and final histopathology. By the higher sensitive Southern-blot on RT-PCR method, RET rearrangements were present in 36% of papillary thyroid carcinomas (RET/PTC-1, 12%; RET/PTC-3, 20%; both, 4%) and of 13.3% of benign nodules. By means of RT-PCR only, RET rearrangements were disclosed only in 14.3% of PTC and in 3.6% of benign nodules. No significant correlation was found between RET rearrangements and clinicopathological features of patients. These results indicate that molecular testing of thyroid nodules for RET/PTC must take into account of its high prevalence in benign nodules, inducing false positive diagnoses when the highly sensitive assay Southern-blot on RT-PCR is used. Its searching by means of RT-PCR only, has a specificity superior of conventional cytology and can be used to refine inconclusive FNAC.

Growing thyroid nodules with benign histology and RET rearrangement.

Some benign thyroid nodules are stationary in size over time while others grow progressively, indicating that there is a broad individual variability within benign nodules. To date, it is very difficult to predict if a benign thyroid nodule will grow in size and which will be its trend over time. While BRAF(V600E) is a highly specific marker of thyroid cancer, RET rearrangements have been disclosed also in non malignant thyroid lesions and their biological significance is debated. We compared the clinical history of three histologically benign thyroid nodules harboring RET rearrangements with that of 6 benign nodules bearing wild type RET. The nodules negative for RET rearrangements were followed for 10 years by ultrasonographic evaluation, showing a slow, constant enlargement. Three patients with benign nodules diagnosed at FNAC, were followed for 11, 9 and 7 years by annual ultrasonographic evaluation. After several years of latency, the nodules had an unexpected and gradual increase in their dimensions, reaching a large final size. A second FNAC confirmed the previous cytologic diagnosis of benign lesion. Because of the increasing size of the nodules, the patients were advised to surgery. Before undergoing thyroidectomy, we performed molecular diagnostic tests that revealed the absence of BRAF(V600E) and the presence of RET/PTC-1 in one nodule and RET/PTC-3 in the two others. Despite the presence of this oncogene, the samples were histologically classified as benign hyperplastic nodules. These findings lead us to speculate that histologically benign hyperplastic thyroid nodules containing RET rearrangements might represent a subgroup of nodules with a rapid size increase.

Combined analysis of galectin-3 and BRAFV600E improves the accuracy of fine-needle aspiration biopsy with cytological findings suspicious for papillary thyroid carcinoma.

Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAFV600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAFV600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAFV600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAFV600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAFV600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAFV600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.

Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA).

OBJECTIVE: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. DESIGN AND METHODS: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. RESULTS: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis. CONCLUSIONS: BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.

The primary occurrence of BRAF(V600E) is a rare clonal event in papillary thyroid carcinoma.

CONTEXT: BRAF(V600E) is considered a primary event, a negative prognostic marker, and a site for pharmacological intervention in papillary thyroid carcinoma (PTC). We asked whether BRAF(V600E) can occur as a subclonal event in PTC and whether this and other oncogenes can coexist in the same tumor. STUDY DESIGN: We determined by pyrosequencing the percentage of mutant BRAF, NRAS, and KRAS alleles in a series of conventional PTC. We also analyzed the BRAF mutation status in PTC cell clones in culture. RESULTS: BRAF(V600E) alleles were present in 41 of 72 PTC (56.9%) in the range 44.7 to 5.1% of total BRAF alleles. In four PTC samples, mutant BRAF alleles were about 50%, being therefore compatible with a clonal heterozygous mutation. In 27 PTC samples, BRAF(V600E) alleles were in the range of 25 to 5.1%. This finding was confirmed after exclusion of the presence of a large contamination by lymphoreticular cells and by the analysis of PTC cells selected by laser capture. Analysis of clones derived from a single cell confirmed the presence of two distinct PTC populations with wild-type or mutated BRAF. Simultaneous subclonal BRAF and KRAS mutations were demonstrated in two PTC. CONCLUSIONS: These data demonstrate that clonal BRAF(V600E) is a rare occurrence in PTC, although frequently this cancer consists of a mixture of tumor cells with wild-type and mutant BRAF. These results suggest that BRAF mutation in PTC is a later subclonal event, its intratumoral heterogeneity may hamper the efficacy of targeted pharmacotherapy, and its association with a more aggressive disease should be reevaluated.

RET/PTC rearrangement in benign and malignant thyroid diseases: a clinical standpoint.

Cytological examination of fine needle aspiration biopsy is the primary means for distinguishing benign from malignant nodules. However, as inconclusive cytology is very frequent, the introduction of molecular markers in the preoperative diagnosis of thyroid nodules has been proposed in recent years. In this article, we review the clinical implications of preoperative detection of rearrangements of the RET gene (RET/papillary thyroid carcinoma (PTC)) in thyroid nodules. The prevalence of RET/PTC in PTC depends on the histological subtypes, geographical factors, radiation exposure, and detection method. Initially, RET/PTC was considered an exclusive PTC hallmark and later it was also found sporadically in benign thyroid lesions. More recently, the very sensitive detection methods, interphase fluorescence in situ hybridization (FISH) and Southern blot on RT-PCR amplicons, demonstrated that the oligoclonal occurrence of RET rearrangement in benign thyroid lesions is not a rare event and suggested that it could be associated with a faster enlargement in benign nodules. For this reason, RET/PTC cannot be considered as an absolute marker of PTC, and its diagnostic application must be limited to assays able to distinguish between clonal and oligoclonal expression. Detection of RET/PTC by quantitative assays will be useful for diagnostic purposes in cytology specimens when a precise cutoff will be fixed in a clinical setting. Until that time, less sensitive RET/PTC detection methods and FISH analysis remain the most appropriate means to refine inconclusive cytology. Future studies with a long follow-up will further clarify the clinical significance of low level of RET rearrangements in benign nodules.

BRAF mutation in cytology samples as a diagnostic tool for papillary thyroid carcinoma.

INTRODUCTION: Thyroid cancer is a rare disease that needs to be differentiated from the more frequent benign nodular goiter. The current, primary technique for distinguishing between benign and malignant nodules is by a fine-needle biopsy (FNB) cytological examination. This type of examination, unfortunately, often provides inconclusive results, and in recent years the introduction of molecular markers for the preoperative diagnosis of thyroid nodules has been proposed. AREAS COVERED: This review covers current and emerging research in the diagnostic application of the BRAF mutation in papillary thyroid carcinomas. It considers the available literature related to the usefulness of preoperative BRAF mutation analysis as a diagnostic tool to refine inconclusive cytology. It also considers the available techniques used to detect this specific mutation. EXPERT OPINION: Many effective methods are now available to detect BRAF mutation in FNB material. Thanks to its high specificity, this genetic alteration is now considered a useful diagnostic marker for patients who have indeterminate thyroid nodule cytology and is a useful tool for thyroid nodule management despite its low sensitivity limiting its application. The authors believe that, in the future, the screening of genetic alterations will enter standard clinical practice as an adjunctive tool to conventional cytology, and larger studies will provide a better definition of the best, most cost-effective combinations of markers and methods.

High growth rate of benign thyroid nodules bearing RET/PTC rearrangements.

CONTEXT: Benign thyroid nodules display a broad range of behaviors from a stationary size to a progressive growth. The RET/PTC oncogene has been documented in a fraction of benign thyroid nodules, besides papillary thyroid carcinomas, and it might therefore influence their growth. OBJECTIVE: The aim of the present work was to evaluate whether RET/PTC in benign thyroid nodules associates with a different nodular growth rate. STUDY DESIGN: In this prospective multicentric study, 125 subjects with benign nodules were included. RET rearrangements were analyzed in cytology samples; clinical and ultrasonographic nodule characteristics were assessed at the start and at the end of the study. RESULTS: RET/PTC was present in 19 nodules. The difference between the mean baseline nodular volume of the RET/PTC- and RET/PTC+ nodules was not significant. After 36 months of follow-up, the RET/PTC+ group (n = 16) reached a volume higher than the RET/PTC- group (n = 90) (5.04 ± 2.67 vs. 3.04 ± 2.26 ml; P = 0.0028). We calculated the monthly change of nodule volumes as a percentage of baseline. After a mean follow-up of 36.6 months, the monthly volume increase of nodules bearing a RET rearrangement was 4.3-fold that of nodules with wild-type RET (1.83 ± 1.2 vs. 0.43 ± 1.0% of baseline volume; P < 0.0001). CONCLUSIONS: Benign thyroid nodules bearing RET rearrangements grow more rapidly than those with wild-type RET. Searching for RET rearrangements in benign thyroid nodules might be useful to the clinician in choosing the more appropriate and timely therapeutic option.

The Ca2+-calmodulin-dependent kinase II is activated in papillary thyroid carcinoma (PTC) and mediates cell proliferation stimulated by RET/PTC.

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRaf(V600E), CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRaf(V600E), or Ras(V12) induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca(2+)/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRaf(V600E), oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.

Detection of RET/PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20-40% of cases the FNAB result is uncertain. OBJECTIVE: We investigated whether a search for the oncogenes RET/PTC, TRK and BRAF(V600E) in thyroid aspirates could refine an uncertain diagnosis. PATIENTS AND METHODS: A total of 132 thyroid aspirates, including colloid nodules, inadequate samplings, indeterminate and suspicious for malignancy were analysed by reverse transcription polymerase chain reaction (RT-PCR) and mutant allele-specific amplification techniques for the presence of oncogenes. RESULTS: No oncogenes were detected in 48 colloid nodules, 46 inadequate and 19 indeterminate FNABs, then confirmed to be benign at histology. No oncogenes were detected in one follicular thyroid cancer (FTC) with indeterminate cytology. Five out of six papillary thyroid cancers (83%) with FNAB suspicious for malignancy were correctly diagnosed by the presence of oncogenes. Among these, four (67%) contained the BRAF mutation and one (17%) contained RET/PTC-3. On final analysis, no false-positive results were reported in 131 samples and five out of seven carcinomas (71%) were correctly diagnosed. The finding of oncogenes in FNAB specimens suspicious for malignancy guided the extent of surgical resection, changing the surgery from diagnostic to therapeutic in five cases. CONCLUSIONS: Detection of RET/PTC, TRK and BRAF(V600E) in FNAB specimens is proposed as a diagnostic adjunctive tool in the evaluation of thyroid nodules with suspicious cytological findings.

The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.

The alae, longitudinal ridges of the lateral cuticle, are the most visible specialization of the Caenorhabditis elegans surface. They are present only in L1 and dauer larvae and in adults. Little is known about the mechanisms through which at the appropriate stages secretion of cuticle components by the seam cells results in the formation of the alae. Here we show that three proteins, each containing a Zona Pellucida domain (ZP), are components of the cuticle necessary for larval alae development: CUT-1 and CUT-5 in dauer larvae and CUT-3 and CUT-5 in L1s. Transcriptional regulation of the corresponding genes contributes to the stage-specific role of these proteins. Larvae with reduced cut-1, cut-3 or cut-5 function not only lack alae but are also larger in diameter due to an increase in the width of the lateral cuticle. We propose a model in which reduction of the body diameter, which occurs in normal L1 and dauer larvae, is the result of a dorso-ventral shrinking of the internal layer of the lateral cuticle and formation of the alae results from the folding of the external layer of the lateral cuticle over the reduced, internal one. Alae of adults appear to form through a different mechanism.