A receptor-antibody hybrid hampering MET-driven metastatic spread.

BACKGROUND: The receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition. METHODS: In this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by 'head-to-tail' or 'tail-to-head' fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant 'ad-hoc' engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope. RESULTS: The hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions. CONCLUSION: These results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis.

Poorly differentiated thyroid carcinoma of childhood and adolescence: a distinct entity characterized by DICER1 mutations.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

Characterization of Activating Mutations of the MEK1 Gene in Papillary Thyroid Carcinomas.

Background: Genetic alterations activating the mitogen-activated protein kinase (MAPK) signaling pathway, most commonly BRAF and RAS mutations, are common in papillary thyroid carcinoma (PTC). Somatic mutations of the MEK gene, also known as mitogen-activated protein kinase kinase 1 (MAP2K1), coding for a signaling protein downstream of BRAF, have been found in several cancer types. The goal of this study was to investigate if functional MEK1 mutations occur in thyroid cancer (TC). Methods: We analyzed MEK1 mutation status in a series of 101 PTCs lacking other known driver mutations using Sanger sequencing and targeted next-generation sequencing. In addition, 64 follicular and Hürthle cell carcinomas and 32 follicular adenomas were studied. The occurrence of MEK1 mutations was evaluated using another series of thyroid tumors studied by targeted next-generation sequencing. Western blot and RNA-seq analyses were performed on selected tumors. Results: We detected MEK1 mutations in 2/101 (2%) PTCs that otherwise had no known genetic alterations, in 0/64 follicular and Hürthle cell carcinomas, and in 0/32 follicular adenomas. Two positive tumors carried the same in-frame deletion p.L98_I103del; K104I (c.292_309del18; c.311A>T) located in exon 3 of the gene. One additional MEK1 mutation was identified following routine molecular tumor profiling. The tumor had an in-frame deletion p.I99_K104del (c.294_311del18) also located in exon 3. Western blot analysis of one of the tumors showed activation of the MAPK pathway. Using RNA-seq analysis to evaluate changes in gene expression, these tumors were RAS-like and showed a high thyroid differentiation score. Phenotypically, the MEK1-positive PTCs were all encapsulated and had a predominantly or exclusively follicular architecture, being diagnosed as a classic papillary type with a significant follicular pattern ( × 2) or follicular variant PTC ( × 1). Follow-up was available for 2 patients, with no evidence of disease found 2 and 10 years postsurgery. Conclusions: In this study, we report the occurrence of functional MEK1 mutations in PTC. All mutations are in-frame deletions in exon 3 of MEK1, representing another mechanism of activation of the MAPK pathway in papillary carcinomas with a predominantly follicular growth pattern.

TERT promoter mutations and their correlation with BRAF and RAS mutations in a consecutive cohort of 145 thyroid cancer cases.

Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy and accounts for ~80% of thyroid carcinomas in adults and 90% in children. Risk stratification is important for identifying patients at higher risk and, for this reason, recent advances in molecular genetics of thyroid cancer can be applied to provide novel biomarkers useful in understanding tumor behavior. B-Raf proto-oncogene, serine/threonine kinase (BRAF) and rat sarcoma (RAS) mutations have been widely studied and appear to have an important role in thyroid tumorigenesis. Somatic telomerase reverse transcriptase (TERT) promoter mutations have been recently identified in several types of malignant tumors, including thyroid neoplasia; however, the actual role of TERT mutations in thyroid tumorigenesis is still under debate. In the present study, the mutational status of BRAF, RAS and TERT was analyzed in order to elucidate the roles of these genes in thyroid tumorigenesis. The TERT mutational analysis was also correlated with an immunohistochemical study of TERT protein expression. According to the literature, our data provide evidence of the BRAF and RAS roles in thyroid tumorigenesis, supporting an association between BRAF (V600E) mutations and the more aggressive clinical and pathological features of thyroid tumors. By contrast, TERT mutations were not significantly associated with any clinical parameters; therefore, its role in initial tumorigenesis should be further investigated.

KIF5B/RET Rearrangement in a Carcinoma of the Thyroid Gland: A Case Report of a Fatal Disease.

Background: The diffuse sclerosing variant of papillary thyroid cancer (DSV-PTC) is a rare variant of papillary thyroid cancer (PTC) with different clinicopathological features compared with conventional PTC. Case: An advanced DSV-PTC was diagnosed in a 39-year-old man. The radioiodine posttherapeutic whole-body-scan showed only an uptake in the central neck, whereas the computerized tomography showed multiple latero-cervical and mediastinum lymph node metastases, a single and spiculated lung lesion and multiple bilateral cerebellum metastases. The patient died after 6 months from the initial diagnosis. The histological revision of the thyroid tumor confirmed the diagnosis of DSV-PTC, and its molecular analysis revealed a KIF5B/RET rearrangement that, until now, was described only in a minority of lung adenocarcinoma. Other 18 cases of DSV-PTC were then studied for the presence of KIF5B/RET rearrangement, but all of them were negative. Conclusions: This was a case of DSV-PTC positive for KIF5B/RET rearrangement, but considering that this alteration has been described only in lung adenocarcinoma and that the clinical course was more typical of lung carcinoma, we cannot completely rule out the possibility that this was a metastatic lesion from a lung tumor mimicking a DSV-PTC. As an alternative, we can also hypothesize that this was a case of fusion of two tumoral tissues deriving from a DSV-PTC and a metastasis of a KIF5B/RET positive lung adenocarcinoma. The question of whether the molecular findings, particularly when specifically reported only in some subtypes of human tumors, can overcome the morphological diagnosis is a matter of discussion.

Differences in miRNA expression profiles between wild-type and mutated NIFTPs.

Noninvasive encapsulated follicular variants of papillary thyroid carcinomas have been recently reclassified as noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs). NIFTPs exhibit a behavior that is very close to that of follicular adenomas but different from the infiltrative and invasive follicular variants of papillary thyroid carcinomas (FVPTCs). The importance of miRNAs to carcinogenesis has been reported in recent years. miRNAs seem to be promising diagnostic and prognostic molecular markers for thyroid cancer, and the combination of miRNA expression and mutational status might improve cytological diagnosis. The aim of the present study was to evaluate the miRNA expression profile in wild-type, RAS- or BRAF-mutated NIFTPs, infiltrative and invasive FVPTCs, and follicular adenomas using the nCounter miRNA Expression assay (NanoString Technologies). To identify the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) molecular pathways associated with deregulated miRNAs, we used the union of pathways option in DNA Intelligent Analysis (DIANA) miRPath software. We have shown that the miRNA expression profiles of wild-type and mutated NIFTPs could be different. The expression profile of wild-type NIFTPs seems comparable to that of follicular adenomas, whereas mutated NIFTPs have an expression profile similar to that of infiltrative and invasive FVPTCs. The upregulation of 4 miRNAs (miR-221-5p, miR-221-3p, miR-222-3p, miR-146b-5p) and the downregulation of 8 miRNAs (miR-181a-3p, miR-28-5p, miR-363-3p, miR-342-3p, miR-1285-5p, miR-152-3p, miR-25-3p, miR-30e-3) in mutated NIFTPs compared to wild-type ones suggest a potential invasive-like phenotype by deregulating the specific pathways involved in cell adhesion and cell migration (Hippo signaling pathway, ECM-receptor interaction, adherens junction, regulation of actin cytoskeleton, fatty acid biosynthesis and metabolism).

miRNA expression profiling of 'noninvasive follicular thyroid neoplasms with papillary-like nuclear features' compared with adenomas and infiltrative follicular variants of papillary thyroid carcinomas.

Follicular variants of papillary thyroid carcinoma include encapsulated (with or without capsular/vascular invasion) and infiltrative forms, which have different clinical behaviors. The encapsulated forms that lack capsular invasion have an indolent clinical behavior that is similar to benign lesions; therefore, they were recently reclassified as 'noninvasive follicular thyroid neoplasms with papillary-like nuclear features' (NIFTPs). Because NIFTPs have nuclear features of papillary carcinomas, distinguishing between NIFTPs and infiltrative follicular variant of papillary thyroid carcinoma is almost impossible with cytological examination. The aim of this study is to determine whether miRNA expression profiles may help distinguish between NIFTPs versus follicular adenomas and infiltrative follicular variant of papillary thyroid carcinomas. The expression profiling of 798 miRNAs was tested in 54 thyroid tumors, including 18 follicular adenomas, 19 NIFTPs and 17 infiltrative follicular variant of papillary thyroid carcinomas, using nCounter Nanostring. We found that miR-146-5p, miR-221-5p, miR-222-3p, miR-30e-3p, and miR-152-3p could discriminate between benign and malignant lesions with a very high level of significance (P-value<0.001). High expression levels of miR-146-5p, miR-199a-5p, miR-199b-5p, miR-1285-5p, miR-1915-3p, and miR-4516, and low miR-148b-3p expression were associated with infiltrative growth of follicular variant of papillary thyroid carcinomas. Interestingly, miR-152-3p, miR-185-5p, and miR-574-3p were significantly downregulated in NIFTPs compared with follicular adenomas, whereas miR-10a-5p and miR-320e can discriminate between NIFTPs and infiltrative forms of follicular variant of papillary thyroid carcinomas. In conclusion, a panel of these markers could have high diagnostic potential as well as could be applied to presurgical fine-needle aspiration, especially for lesions classified as indeterminate thyroid nodules.

Role of YAP-1 in Thyroid Tumor Progression and Outcome.

CONTEXT: Yes-associated protein-1 (YAP-1) is a player of the Hippo pathway and is involved in regulating cell proliferation. YAP-1 is overexpressed in papillary and anaplastic thyroid cancers. However, a correlation between YAP-1 expression and outcome in thyroid carcinoma has not been conclusively demonstrated. OBJECTIVE: This study was designed to clarify whether YAP-1 may be considered a marker of worse prognosis and outcome in thyroid cancer. DESIGN: A large series of cases of thyroid cancer with a long follow-up were investigated for YAP-1 expression. SETTING: The study was carried out in the Pathology section of a referral Italian center for Endocrine Surgery and Endocrinology. PATIENTS OR OTHER PARTICIPANTS: The study included a consecutive series of 105 patients who underwent thyroidectomy from 1985 to 1992. The mean follow-up was 15 years. For all patients, clinicopathologic features were considered. All patients completed the study.The study also included a consecutive series of 52 patients who underwent thyroidectomy from 2012 to 2013 in order to analyze more deeply the correlation of YAP-1expression with BRAF mutation. INTERVENTION: The 105 thyroid tumors were immunohistochemically investigated for YAP-1 expression. OUTCOME MEASURES: We expected a correlation between YAP-1 expression and worse prognosis. RESULTS: Among 105 tumors, 77 scored positive for YAP-1 expression, of which 68 papillary thyroid carcinomas and 9 anaplastic thyroid carcinomas were YAP-1 positive. The correlation of YAP-1 expression with clinicopathologic characteristics was significant for the absence of a tumoral capsule, gender, and extrathyroid invasion.Interestingly, significant correlations were found between YAP-1 and both persistence of disease and death from carcinoma. CONCLUSIONS: The data show an association of YAP-1 expression with worse clinicopathologic features of thyroid tumors that seem to have a specific impact on outcome.

Low Elasticity of Thyroid Nodules on Ultrasound Elastography Is Correlated with Malignancy, Degree of Fibrosis, and High Expression of Galectin-3 and Fibronectin-1.

BACKGROUND: Thyroid ultrasound (US) elastography provides an estimation of tissue stiffness and is helpful to differentiate malignant from benign lesions. Tissue proprieties and molecules causing stiffness are not established. The aim of the study was to correlate US elastography findings with tissue properties in thyroid nodules. METHODS: A total of 115 thyroid nodules from 112 patients who underwent surgery for the presence of Thy 3 (indeterminate) cytology (n = 67), Thy 4-5 (suspicious-indicative of carcinoma) cytology (n = 47), or large goiter in the presence of Thy 2 cytology (n = 1) and suspicious US features were examined by US elastography. Tissues obtained after surgery were characterized for cell number, microvessel density, fibrosis, and expression of galectin-3 (Gal-3) and fibronectin-1 (FN-1). RESULTS: Low elasticity on qualitative US elastography (LoEl) was found in 66 nodules (one benign and 65 carcinomas); high elasticity (HiEl) was found in 49 nodules (46 benign and three carcinomas; p < 0.0001). Quantitative analysis, performed in 24 nodules and expressed as elastic ratio between the strain of the nodule and that of the surrounding thyroid parenchyma, showed a mean of 1.90 (interquartile range [IQR] 1.18-2.77) in 14 nodules with LoEl, and a mean of 1.01 (IQR 0.91-1.10) in 10 nodules with HiEl (p = 0.002). Stiffness did not correlate with cell number and was inversely correlated with microvessel density. Fibrosis was higher in nodules with LoEl than in those with HiEl (p = 0.009) and in carcinomas than in benign nodules (p = 0.02). Fibrosis was higher in nodules with high expression of Gal-3 (p < 0.001) and FN-1 (p = 0.004). Fibrosis and expression of Gal-3 and FN-1 were higher in the classic compared with the follicular variant of papillary thyroid carcinoma and lower in follicular adenomas. CONCLUSIONS: Low elasticity at US elastography is highly correlated with malignancy. Nodule stiffness is correlated with fibrosis and expression of Gal-3 and FN-1. These features are more evident in the classic than in the follicular variant of papillary thyroid carcinoma.

EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large­scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix­assisted laser desorption ionization­time of flight (MALDI­TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI­TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI­TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI­TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.

Role of gene expression profiling in defining indeterminate thyroid nodules in addition to BRAF analysis.

Fine-needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, 15% to 30% of aspirations yield indeterminate cytologic findings. Because the assessment of BRAF mutations seems to improve the diagnostic accuracy, this study evaluated BRAF mutations with Sanger sequencing and real-time methods in 650 consecutive thyroid aspirates. In addition, the expression of a large number of genes involved in basement membrane remodeling, extracellular matrix proteolysis, and cell adhesion was studied in both benign and malignant nodules to identify new diagnostic tools. In this prospective series, despite the use of a very sensitive BRAF mutational testing method, the frequency of a BRAF alteration being identified in indeterminate FNA samples was 3 of 68. Expression analysis revealed several genes that were differentially expressed between benign and malignant nodules (transforming growth factor, cadherin 1, collagen α1, catenin α1, integrin α3, and fibronectin 1 [FN1]), between follicular adenomas and follicular variant of papillary thyroid carcinoma (FN1, laminin γ1, integrin ß2, connective tissue growth factor, catenin δ1, and integrin αV), and between BRAF-wild-type and BRAF-mutated papillary thyroid carcinomas (TIMP metallopeptidase inhibitor 1; catenin α1; secreted phosphoprotein 1; FN1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; and selectin L). These data were partially confirmed with real-time polymerase chain reaction analysis and immunohistochemistry. When the cost/benefit ratio of the procedures was taken into account, BRAF mutational testing failed to increase diagnostic accuracy in cytologically indeterminate nodules. However, the additional analysis of the expression of specific molecular markers could have possible utility as a diagnostic tool, although further evidence based on a large series of samples is needed before definitive conclusions can be drawn. Cancer Cytopathol 2016;124:340-9. © 2015 American Cancer Society.

Digital gene expression profiling of a series of cytologically indeterminate thyroid nodules.

BACKGROUND: Fine-needle aspiration cytology (FNAC) has been widely accepted as the most crucial step in the preoperative assessment of thyroid nodules. Testing for the expression of specific genes should improve the accuracy of FNAC diagnosis, especially when it is performed in samples with indeterminate cytology. METHODS: In total, 69 consecutive FNACs that had both cytologic and histologic diagnoses were collected, and expression levels of 34 genes were determined in RNA extracted from FNAC cells by using a custom digital mRNA counting assay. A supervised k-nearest neighbor (K-nn) learning approach was used to build a 2-class prediction model based on a subset of 27 benign and 26 malignant FNAC samples. Then, the K-nn models were used to classify the 16 indeterminate FNAC samples. RESULTS: Malignant and benign thyroid nodules had different gene expression profiles. The K-nn approach was able to correctly classify 10 FNAC samples as benign, whereas only 1 sample was grouped in the malignant class. Two malignant FNAC samples were incorrectly classified as benign, and 3 of 16 samples were unclassified. CONCLUSIONS: Although the current data will require further confirmation in a larger number of cases, the preliminary results indicate that testing for specific gene expression appears to be useful for distinguishing between benign and malignant lesions. The results from this study indicate that, in indeterminate FNAC samples, testing for cancer-specific gene expression signatures, together with mutational analyses, could improve diagnostic accuracy for patients with thyroid nodules.

E-selectin expression and BRAF status in papillary thyroid carcinomas: Correlation with clinicopathologic features.

BACKGROUND: Cell adhesion molecules, represented by the immunoglobulin family and selectins, play an important role in the progression of cancer. A correlation between selectins and tumor aggressiveness has been demonstrated in several reports. METHODS: Eighty-eight patients (mean age, 41.0 ± 14 years) with papillary thyroid carcinoma (conventional variant and sized approximately 20 mm) were divided in 2 groups: 41 with encapsulated tumors and 47 with tumors with extrathyroidal extension. E-selectin expression was evaluated by immunohistochemical staining and semiquantitative real-time reverse-transcription polymerase chain reaction and normalized by calculating the z-score (positive: value above the population mean; negative: below the mean). RESULTS: Lymph node metastasis (LNM) was found in 2 of 41 encapsulated tumors (4.8%) and in 19 of 47 tumors (40.4%) with extrathyroidal extension. BRAF mutation was present in 21 encapsulated tumors (51.2%) and in 31 tumors with extrathyroidal extension (65.9%). The mean E-selectin z-score was -0.32 for encapsulated tumors and 0.28 for tumors with extrathyroidal extension. E-selectin expression correlates with neoplastic infiltration (P = .04), the American Joint Commission on Cancer stage (P = .02), and BRAF mutation (P = .03). CONCLUSION: E-selectin overexpression in association with BRAF mutation status could promote a more aggressive phenotype in papillary thyroid carcinoma.

ALK rearrangement in a large series of consecutive non-small cell lung cancers: comparison between a new immunohistochemical approach and fluorescence in situ hybridization for the screening of patients eligible for crizotinib treatment.

CONTEXT: Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. OBJECTIVE: To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. DESIGN: We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. RESULTS: Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. CONCLUSIONS: Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.

Molecular characterization of 54 cases of false-negative fine-needle aspiration among 1347 papillary thyroid carcinomas.

BACKGROUND: Fine-needle aspiration (FNA) has been widely accepted as the most crucial step in the preoperative assessment of thyroid nodules, but the false-negative rates are generally reported to be between 3.6% and 10.2%. To lower the overall incidence of this false-negative testing, new reporting systems encourage the molecular testing of thyroid nodules. However, to the authors' knowledge, the role of molecular testing in false-negative FNA has not yet been evaluated. METHODS: In total, 1347 consecutive papillary thyroid carcinomas (PTCs) with both cytological and histological diagnoses were collected from the same center. A blinded revision of the false-negative cases was performed. An analysis of the BRAF and Ras genes in the false-negative cases was then performed. RESULTS: The false-negative rate at the time of primary FNA diagnosis was 4.8% (65 of 1347 cases). False-negative cases were 15 follicular variant PTCs, 2 classical variant, and 1 solid variant that lacked peculiar PTC cytomorphological features. Adequate cellular material for molecular analysis was available only in 54 of the 65 false-negative cases. Mutations were found in 6 cases (11%), and Ras alterations were present in 16 cases (29.6%). The addition of molecular analysis decreased the false-negative rate to 0.4% (5 of 1347 cases). CONCLUSIONS: The results of the current study confirm the feasibility of BRAF and Ras analysis in routine FNA. However, when the false-negative FNA rate is low, the cost-benefit analysis of the detection of BRAF and Ras mutations should be carefully evaluated. Consequently, the authors suggest that preoperative molecular assessment could be helpful for benign nodules, but only in the presence of clinical suspicion of malignancy.

FoxP3 expression in papillary thyroid carcinoma: a possible resistance biomarker to iodine 131 treatment.

BACKGROUND: The forkhead transcription factor FoxP3 plays an important role in regulatory T cell (Treg) functions. Tregs are critical in maintaining immunologic tolerance. It has been shown that vaccination against FoxP3-expressing cells is associated with enhancement of tumor immunity. Tregs appear to be increased in blood and in the tumor microenvironment of patients with different cancer types. Tumor cells themselves can express FoxP3. The present study investigates the possible role of FoxP3 expression in a series of human papillary thyroid cancers with a mean follow-up time of 15 years. METHODS: One hundred five cases of papillary thyroid carcinoma (PTC) were investigated, and FoxP3 expression was evaluated in both tumor cells and tumor-associated infiltrates. For all patients, clinical/pathologic features were considered and the results analyzed by statistical tests. RESULTS: Of the 105 PTC cases, 45 (43%) scored FoxP3-positive and 60 (57%) were negative. FoxP3 staining was localized predominantly in the cytoplasm of tumor cells. In some cases, both nuclear and cytoplasmic staining was seen in infiltrating cells. FoxP3 expression in tumor cells was correlated with the presence of extrathyroid invasion (p=0.04) and distant metastasis (p=0.04), but not with overall survival. Interestingly, FoxP3 expression in neoplastic cells was significantly associated with a resistance phenotype to radioiodine treatment (p=0.041). CONCLUSIONS: The data show an association of FoxP3 expression with features of PTC that seem to have a specific impact on radioiodine sensitivity.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. METHODS: We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. RESULTS: Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. CONCLUSIONS: We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

Antiproliferative and proapoptotic activity of sunitinib on endothelial and anaplastic thyroid cancer cells via inhibition of Akt and ERK1/2 phosphorylation and by down-regulation of cyclin-D1.

CONTEXT: Recent experimental evidence suggests a rationale for the use of multitarget tyrosine kinase inhibitors for the treatment of thyroid cancers. Sunitinib showed promising preliminary results against anaplastic thyroid cancer (ATC), and it has been used for some patients who are ineligible for clinical trials. OBJECTIVES: The aims of this study were to investigate the in vitro and in vivo activity of sunitinib on ATC and on microvascular endothelial cells and the molecular mechanism for the observed sunitinib activity. METHODS: Proliferation and apoptotic assays were performed on human dermal microvascular endothelial and on BRAF- or H-ras-mutated ATC cells (8305C and FB3, respectively) after in vitro exposure to sunitinib for 72 hours. Vascular endothelial growth factor receptor-2, epithelial growth factor receptor, ERK1/2, and Akt phosphorylation was quantified by ELISA and Western blot. Cyclin-D1 mRNA expression was evaluated by real-time PCR, and cyclin-D1 intracellular concentrations were measured by ELISA. 8305C tumor xenografts in nude mice were treated with sunitinib at 50 mg/kg/d (i.p.). RESULTS: Antiproliferative and proapoptotic activity of sunitinib was observed in both endothelial and ATC cells. Phospho-vascular endothelial growth factor receptor-2 levels significantly decreased after sunitinib treatment in activated endothelial cells. Phospho-epidermal growth factor receptor, ERK1/2, and Akt phosphorylation was significantly inhibited by sunitinib treatment in endothelial and cancer cells, and cyclin-D1 mRNA and protein expression was inhibited. Sunitinib administration in vivo caused significant inhibition of tumor growth (P < .05). CONCLUSIONS: Sunitinib is active in vitro and in vivo against activated endothelial and ATC cells via the inhibition of Akt and ERK1/2 phosphorylation and through the down-regulation of cyclin-D1.

KIF5B/RET fusion gene analysis in a selected series of cytological specimens of EGFR, KRAS and EML4-ALK wild-type adenocarcinomas of the lung.

A new RET fusion gene has been recently described in a subset of non-small cell lung cancer (NSCLC) identified by specific clinico-pathologic characteristics. This transforming gene arise from the fusion of KIF5B and the RET proto-oncogene, and it is mutually exclusive with EGFR, KRAS and EML4/ALK alterations. For this reason it could represent a putative target for specific inhibitory drugs and its evaluation could be necessary in the future daily molecular characterization of NSCLCs. One of the major challenge in diagnostic molecular pathology is to optimize genotyping tests with the minimally invasive techniques used to acquire diagnostic tumor tissue or cells. This is a significant relevant issue for approximately 60% of NSCLC patients presenting with unresectable disease, where the only pathologic materials available for diagnostic use are small biopsy or cytological specimens. Thus, the aim of this study was to verify the possibility to use RNA purified from cytological specimens to perform KIF5B/RET gene fusion expression analysis. Accordingly, we looked for the presence of the rearrangement in formalin fixed paraffin embedded tissues (FFPETs) and cytological specimens (CSs) of a selected series of "triple-marker" negative adenocarcinomas. The tests conducted revealed the presence of 1 positive patient for variant 1 of KIF5B/RET among the 49 analyzed. The presence of this fusion transcript was found in both FFPET and CS of the same patient demonstrating that the RNA obtained from minimally invasive techniques is perfectly suitable for this kind of tests. The presence of the rearrangement was also confirmed by FISH analysis. In conclusion, our findings confirm that the performance of cytology-based molecular testing for KIF5B/RET rearrangements is at least as effective as histology-based analysis, both with regard to the success rate for nucleic acid isolation and the ability to detect gene alterations.

EML4-ALK translocation in both metachronous second primary lung sarcomatoid carcinoma and lung adenocarcinoma: a case report.

The EML4-ALK gene translocation was described in a non small cell lung cancer (NSCLC) subset, with a potent oncogenic activity. It represents one of the newest molecular targets in NSCLC. We report on the case of a metachronous second primary lung sarcomatoid carcinoma after resection of lung adenocarcinoma both with ALK translocation, in a non-smoking patient. EML4-ALK rearrangement was detected with immunohistochemistry and confirmed with fluorescent in situ hybridization (FISH). To assess the clonal relationship between the two tumors, both adenocarcinoma and sarcomatoid carcinoma were analyzed by array comparative genomic hybridization (aCGH). We observed different genomic profiles suggesting that the tumors arose independently and were thus multiple primaries. To the best of our knowledge, this is the first report concerning the presence of the EML4-ALK fusion gene in a sarcomatoid carcinoma of the lung. Crizotinib, the ALK tyrosine kinase inhibitor, is highly effective in ALK-rearranged NSCLC; therefore, it may be imperative to identify all NSCLC that harbor ALK translocations in the near future. Starting from our evidence, tumors with sarcomatoid histology may need to be screened for the presence of EML4-ALK rearrangement.