Moira Ragazzi

TERT promoter mutations are associated with distant metastases in Papillary Thyroid Carcinoma

OBJECTIVE Transcriptional activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene were reported at high frequency in aggressive poorly differentiated and anaplastic thyroid cancers. By contrast, the relevance of these mutations in the metastatic behavior of well-differentiated thyroid cancer is still to be defined. The aim of this work was to investigate the frequency of TERT promoter mutations in a remarkable cohort of well-differentiated papillary thyroid carcinoma that developed distant metastases (DM-PTCs) and to establish whether these mutations may be predictive of metastatic behavior. DESIGN We analyzed the frequency of TERT promoter mutations in a group of 43 highly aggressive DM-PTCs. As controls, we analyzed these mutations in a group of 78 PTCs without distant metastases (control-PTCs). The possible correlation between TERT promoter mutations and BRAF V600E mutation was also investigated. METHODS TERT promoter mutational status was evaluated by direct sequencing of the hotspot harboring the C228T and the C250T mutations. RESULTS In the overall cohort of 121 PTCs analyzed, 17% of cases (21/121) carried a mutation in the TERT promoter. Noticeably, 33% of DM-PTCs were mutated in the TERT promoter while only 9% of the control-PTCs showed a mutation in this locus. We also observed a positive association between BRAF V600E and TERT C228T mutations in the cohort of DM-PTCs. CONCLUSIONS These results indicate that TERT promoter mutations are associated with the development of distant metastases in PTCs and may help in predicting aggressive behavior in this type of tumor.

Update on Anaplastic Thyroid Carcinoma: Morphological, Molecular, and Genetic Features of the Most Aggressive Thyroid Cancer

Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer. It shows a wide spectrum of morphological presentations and the diagnosis could be challenging due to its high degree of dedifferentiation. Molecular and genetic features of ATC are widely heterogeneous as well and many efforts have been made to find a common profile in order to clarify its cancerogenetic process. A comprehensive review of the current literature is here performed, focusing on histopathological and genetic features.

BRAFV600E Mutation Does Not Mean Distant Metastasis in Thyroid Papillary Carcinomas

CONTEXT The oncogenic BRAFV600E mutation has been frequently associated with aggressive behavior of papillary thyroid carcinomas. However, controversy exists on the consistency of these data, and very little is known about the relationship between this genetic alteration and the tendency of papillary thyroid carcinoma (PTC) to develop metastasis at distant sites. STUDY DESIGN We analyzed by direct sequencing the frequency of BRAFV600E mutation within a group of 47 highly aggressive PTC, which had developed distant metastases. As control, we analyzed the BRAFV600E mutation in a group of 75 PTC without distant metastases who were disease free for a minimum 7-yr follow-up. RESULTS The BRAFV600E mutation was present in 29.8% of the distantly metastatic PTC, whereas it was detected in about 44.0% of the control tumors. CONCLUSION These results clearly prove that the BRAFV600E mutation is not associated with the development of distant metastases or fatal outcome in PTC and may not predict aggressive behavior in this type of tumor.

Allele Percentage of the BRAF V600E Mutation in Papillary Thyroid Carcinomas and Corresponding Lymph Node Metastases: No Evidence for a Role in Tumor Progression

CONTEXT The relevance of the BRAF V600E mutation in papillary thyroid carcinoma (PTC) as a negative prognostic factor is a subject of intense debate. This mutation has been associated with several clinicopathological features, but the lack of consistency among data does not support its usefulness as marker of tumor aggressiveness and poorer outcome. Due to the genetic heterogeneity of the tumor, both the occurrence and the allele percentage of the BRAF mutation should be considered to unravel this controversy. OBJECTIVE We aimed to evaluate the impact of the BRAF V600E mutation occurrence and the allele percentage on the metastatic process in PTCs. STUDY DESIGN The presence and allele percentage of the BRAF mutation were determined by pyrosequencing in 132 cases of well-differentiated PTCs with (n = 37) or without lymph node metastases (LNMs) (n = 95) and in 40 LNMs matched with 35 PTCs. RESULTS No significant differences were observed in either the occurrence or the allele percentage of V600E mutation between the 2 groups of PTCs with or without LNMs. The LNMs were heterogeneous for the V600E mutation as the primary lesions. CONCLUSIONS In this study, the occurrence and percentage of the BRAF V600E mutated allele was not preferentially associated with the development of metastases and the average mutated allele percentage decreased as the tumor progresses from the primary site to the lymph node metastatic sites. These observations support the need to reevaluate the role of the BRAF V600E mutation as a negative prognostic marker in PTCs.

Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen‐ or fixed‐section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM.

High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas

CONTEXT The homogeneous distribution of BRAF V600E in papillary thyroid carcinoma (PTC) has been called into question by recent reports. These studies claim that BRAF V600E is heterogeneous and is limited to tumor cell subsets in the majority of PTCs. OBJECTIVE The objective of the study was to understand the allele distribution of BRAF V600E by evaluating the percentage of mutated neoplastic cells in a group of PTCs using two different highly sensitive analytical approaches: allele-specific locked nucleic acid PCR and 454 next-generation sequencing targeted to BRAF exon 15. STUDY DESIGN BRAF V600E was investigated using allele-specific locked nucleic acid PCR on 155 consecutive samples of PTC. Mutated cases were reanalyzed by 454 next-generation sequencing and immunohistochemistry. Because the evaluation of genetic heterogeneity in tumor samples can be profoundly biased by contamination with normal cells, all mutation frequency data were normalized to the real amount of neoplastic cells within each tumor. RESULTS Eighty-five of 155 PTCs (54.8%) were BRAF V600E mutated. The distribution of mutated neoplastic cells within the tumor was as follows: greater than 80% in 37 of 85 (43.5%), 30-80% in 39 of 85 (45.9%), and less than 30% in 9 of 85 (10.6%). In most of the PTCs with less than 80% BRAF V600E-positive neoplastic cells, the mutation was present in large neoplastic cell subpopulations. Tumors with less than 30% mutated neoplastic cells were smaller than tumors with a percentage of mutated cells greater than 80% or between 30% and 80% (P < .05). CONCLUSIONS BRAF V600E is heterogeneously distributed in some PTCs. The large BRAF V600E neoplastic cell subpopulations found in mutated cases is consistent with the view that the BRAF V600E is acquired early during PTC development.

Fluorescence confocal microscopy for pathologists

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium–stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists’ viewpoint.

Cadherin-6 promotes EMT and cancer metastasis by restraining autophagy

The transdifferentiation of epithelial cells toward a mesenchymal condition (EMT) is a complex process that allows tumor cells to migrate to ectopic sites. Cadherins are not just structural proteins, but they act as sensors of the surrounding microenvironment and as signaling centers for cellular pathways. However, the molecular mechanisms underlying these signaling functions remain poorly characterized. Cadherin-6 (CDH6) is a type 2 cadherin, which drives EMT during embryonic development and it is aberrantly re-activated in cancer. We recently showed that CDH6 is a TGFβ target and an EMT marker in thyroid cancer, suggesting a role for this protein in the progression of this type of tumor. Papillary thyroid carcinomas (PTCs) are usually indolent lesions. However, metastatic spreading occurs in about 5% of the cases. The identification of molecular markers that could early predict the metastatic potential of these lesions would be strategic to design more tailored approaches and reduce patients overtreatment. In this work, we assessed the role of CDH6 in the metastatic progression of thyroid cancer. We showed that loss of CDH6 expression profoundly changes cellular architecture, alters the inter-cellular interaction modalities and attenuates EMT features in thyroid cancer cells. Using a yeast two-hybrid screening approach, based on a thyroid cancer patients library, we showed that CDH6 directly interacts with GABARAP, BNIP3 and BNIP3L, and that through these interactions CDH6 restrains autophagy and promotes re-organization of mitochondrial network through a DRP1-mediated mechanism. Analysis of the LIR domains suggests that the interaction with the autophagic machinery may be a common feature of many cadherin family members. Finally, the analysis of CDH6 expression in a unique cohort of human PTCs showed that CDH6 expression marks specifically EMT cells. and it is strongly associated with metastatic behavior and worse outcome of PTCs.

Cadherin 6 is a new RUNX2 target in TGF-β signalling pathway.

Modifications in adhesion molecules profile may change the way tumor cells interact with the surrounding microenvironment. The Cadherin family is a large group of transmembrane proteins that dictate the specificity of the cellular interactions. The Cadherin switch that takes place during epithelial-mesenchymal transition (EMT) contributes to loosening the rigid organization of epithelial tissues and to enhancing motility and invasiveness of tumor cells. Recently, we found Cadherin-6 (CDH6, also known as K-CAD) highly expressed in thyroid tumor cells that display mesenchymal features and aggressive phenotype, following the overexpression of the transcriptional regulator Id1. In this work, we explored the possibility that CDH6 is part of the EMT program in thyroid tumors. We demonstrate that CDH6 is a new transforming growth factor-β (TGF-β) target and that its expression is modulated similarly to other EMT mesenchymal markers, both in vitro and in thyroid tumor patients. We show for the first time that CDH6 is expressed in human thyroid carcinomas and that its expression is enhanced at the invasive front of the tumor. Finally, we show that CDH6 is under the control of the transcription factor RUNX2, which we previously described as a crucial mediator of the Id1 pro-invasive function in thyroid tumor cells. Overall, these observations provide novel information on the mechanism of the EMT program in tumor progression and indicate CDH6 as a potential regulator of invasiveness in thyroid tumors.

Inserting ex vivo Fluorescence Confocal Microscopy Perioperatively in Mohs Micrographic Surgery Expedites Bedside Assessment of Excision Margins in Recurrent Basal Cell Carcinoma

Mohs micrographic surgery can be employed in recurrent basal cell carcinoma, although it is a time-consuming technique. Recently, ex vivo fluorescence confocal microscopy (FCM) has been employed to obtain a fast assessment of tumor margins at the bedside. In our case we successfully employed ex vivo FCM to assess the tumor margins and we treated the persistent tumor with intensity-modulated radiation therapy. Our case demonstrates that a multidisciplinary approach is very efficient in managing complex and recurrent tumors and highlights the benefits of FCM as a new technique that can be used in the surgical theater to speed up the entire procedure.