Maria Rosaria Sapio

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.

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.

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.

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.

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.

Are RET/PTC Rearrangements in Benign Thyroid Nodules of Biological Significance?

RET/PTC are chimeric oncogenes generated by the fusion of the catalytic domain of the tyrosine kinase receptor RET to the 50 terminal region of heterologous genes. While initially RET/PTC expression was considered restricted to papillary thyroid cancer (PTC), highly sensitive detection techniques disclosed the presence of this oncogene also in nonmalignant thyroidal diseases like Hashimoto’s thyroiditis, adenomas, and benign colloid nodules (1). To date, the biological significance of RET/PTC in benign thyroid lesions controversial. While it could represent an event in the transition to malignancy, it might affect cell behavior in other ways. Here, we present a patient with a thyroid nodule that was benign on cytology and histopathology. Molecular analysis revealed that it harbored RET/PTC. Because of the juxtaposition of this with a distinctive clinical course, we speculated that RET rearrangements may be associated with more rapid nodular growth. The patient was a 69-year-old woman who we followed at our University Hospital for 12 years. She presented to us in 1998 for a slight increase in volume in the anterior region of the neck. She had been subjected to subtotal thyroidectomy at the age of 40 years with a histological postoperative diagnosis of benign nodular goiter and since then had been on continuous replacement therapy with L-thyroxine. Physical examination of the neck revealed a mobile hard left thyroidal mass about 2 cm of diameter. There was no palpable cervical lymphadenopathy and she had no cervical compressive symptoms. The results of thyroid function tests were in the normal range and thyroid auto-antibodies were absent. Thyroid ultrasonography revealed recidivism of multinodular goiter. The dominant nodule was in the left lobe. It was solid and hypoechoic on ultrasonography with a mean volume of 2.34 mL. Fine-needle aspiration cytology of the nodule was benign and Thy 2 according to cytological classification of the British Thyroid Association. Taking into account of the benign cytology, the absence of compressive symptoms, and the age of the patient, we decided to follow her with annual ultrasonographic evaluation. The nodule size was stable until 2004, but between 2005 and 2009 the nodule rapidly increased in dimension, reaching the final volume of 7.48 mL (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/thy). A second fine-needle aspiration cytology was performed in late 2009, which confirmed the previous cytologic diagnosis of benign lesion. Nevertheless, because of the increasing size of the nodule and the onset of compressive symptoms, we advised surgery for the patient, which she consented to. The thyroidectomy specimen was independently examined by three pathologists who agreed with the diagnosis of benign hyperplastic nodule and excluded the presence of any foci of malignancy or features of lymphocytic thyroiditis (Supplementary Fig. S2). Studies for RET rearrangements by Southern blot on reverse transcriptase–polymerase chain reaction products were performed as previously described (2) on both cytological and histological specimens (Supplementary Table S1). Southern blot clearly showed the presence of RET/PTC1 (Supplementary Fig. S3). The presence of the oncogene was confirmed by dideoxy sequencing of material from the nodule in the thyroidectomy specimen. Although a direct correlation between the rapid size increase of the nodule and the appearance of RET/PTC1 cannot be demonstrated, it is possible that the rapid nodule growth that occurred in this patient was empowered by the presence of RET/PTC1. Interphase fluorescence in situ hybridization analysis has demonstrated that subclonal RET rearrangements are very frequent in PTC (3). Even if RET/PTC is present only in a sub-set of cells, it might influence the entire nodular mass or a large part of it. Chemokines, small cytokines that bind to 7-transmembrane receptors present on the cell surface, activate a kinase cascade leading to activation of mitogenactivated protein kinases and ultimately in cell migration and proliferation. Expression of the chemokines CXCL1 and CXCL10 and their corresponding receptors is induced by RET/PTC, therefore modulating cell proliferation also by an autocrine/paracrine mechanism (4). Therefore, the oncogene RET/PTC might stimulate proliferation of the neighbor cells by a paracrine loop, extending his effect to the oncogenenegative cells. Although to date there is no evidence that the