Cytological and Ultrasound Features of Thyroid Nodules Correlate With Histotypes and Variants of Thyroid Carcinoma.

CONTEXT: Prognosis is excellent for papillary thyroid carcinoma (PTC), noninvasive follicular thyroid neoplasia with papillary-like nuclear features (NIFT-P), and follicular thyroid carcinoma (FTC) but is poor for poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC). Among PTCs, the prognosis is more favorable for follicular (FV-PTC) and classic (CV-PTC) than for tall cell (TCV-PTC), and solid (SV-PTC) variants. OBJECTIVE: To associate histotypes and variants of thyroid carcinoma with ultrasound and cytological features. METHODS: Histology of 1018 benign tumors and 514 PTC (249 CV, 167 FV, 49 TC, 34 SV, and 15 other variants), 52 NIFT-P, 50 FTC, 11 PDTC, and 3 ATC was correlated with fine-needle aspiration biopsy categories (Italian classification: TIR1, TIR2, TIR3A, TIR3B, TIR4, and TIR5) and ultrasound features at the Endocrinology Unit, University Hospital of Pisa. In total, 1117 patients with thyroid nodule(s) who underwent thyroidectomy were included. RESULTS: Of PTC, 36.3% had indeterminate cytology (TIR3A or TIR3B), 56.6% were suspicious for malignancy or malignant (TIR4 or TIR5); 84.0% FTC and 69.3% NIFT-P were TIR3A or TIR3B; 72.5% FV-PTC and 73.6% SV-PTC were TIR3A or TIR3B; 79.9% CV-PTC and 95.9% TCV-PTC were TIR4 or TIR5. The association of a hypoechoic pattern, irregular margins, and no microcalcifications was more frequent in TCV-PTC than in CV-PTC (P = .02, positive predictive value = 38.9%; negative predictive value = 85.5%). CONCLUSION: At cytology, most FTC, NIFT-P, FV-PTC, and SV-PTC were indeterminate, most CV-PTC and TCV-PTC were suspicious for malignancy or malignant. Ultrasound can be helpful in ruling out TCV-PTC.

Dynamic Profiling of the Immune Tumor Microenvironment in Locally Advanced Gastric Cancer Treated with Perioperative Chemotherapy.

INTRODUCTION: In locally advanced gastric cancer (GC), FLOT represents the standard perioperative regimen and combination with immunotherapy is under investigation. However, the role of immune tumor microenvironment (TME) is poorly recognized in this setting. We aimed to study TME characteristics and dynamics during FLOT. METHODS: Paired biopsy (PRE) and surgical (POST) samples of 25 patients treated with FLOT were prospectively analyzed. After collection of clinic-pathological data, NanoString analyses were performed. The primary objective of the study was to assess the changes induced by chemotherapy in POST compared to PRE samples. RESULTS: The unsupervised hierarchical method analysis clearly distinguished PRE and POST samples, even though some cases showed high immune gene expression at baseline. When POST samples were compared with PRE, a differential expression in hyper-expressed gene sets related to cytotoxicity, T-cell functions, complement system, tumor necrosis factor superfamily, cell cycle, and regulation was recognized. Downstaging of the primary tumor (T-regression, measured by pathologic compared to clinical T stage) was the covariate most frequently associated with these changes. Using the immune cell profiling, cases with T-regression reported a significant increase of T, CD8+ T and B cells and a decrease in mast cells, while nonresponders demonstrated an increase of T, B, cytotoxic, and mast cells. CONCLUSION: Our analysis shows that FLOT significantly influences immune TME of GC. While relevant modifications preferentially occur in tumors showing primary tumor regression, response to treatment seems to be associated with a specific immune profile.

Obesity, Hyperfiltration, and Early Kidney Damage: A New Formula for the Estimation of Creatinine Clearance.

CONTEXT: Glomerular hyperfiltration may represent a direct pathogenetic link between obesity and kidney disease. The most widely used methods to estimate creatine clearance such as Cockroft-Gault (CG), Modification of Diet in Renal Disease (MDRD), and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) have not been validated in subjects with obesity. OBJECTIVE: The performance of prediction formulas was compared with measured creatinine clearance (mCrCl) in subjects with obesity. METHODS: The study population included 342 patients with obesity (mean BMI 47.6 kg/m2) without primary kidney disease. A urine collection was performed over 24 hours for measurement of CrCl. RESULTS: mCrCl increased with body weight. The CG formula showed an overestimation at high CrCl, whereas an underestimation resulted from CKD-EPI and MDRD. To improve the accuracy of estimated CrCl (eCrCl), a new CG-based formula was developed:53+0.7×(140-Age)×Weight/(96xSCr)×(0.85iffemale)A cut-off point for BMI of 32 kg/m2 was identified, at which the new formula may be applied to improve eCrCl. CONCLUSION: In patients with obesity the glomerular filtration rate increases with body weight, and it is associated with the presence of albuminuria, suggesting an early kidney injury. We propose a novel formula that improves the accuracy of eCrCl to avoid missed diagnoses of hyperfiltration in patients with obesity.

Outcomes of the Tall-Cell Variant of Papillary Thyroid Carcinoma in Patients with Different Ages: A 17-Year Mono-Institutional Experience.

The tall-cell variant of papillary thyroid carcinoma (TCPTC) is the most common aggressive variant of papillary thyroid carcinoma (PTC) and typically occurs in older patients. In this study, we analyzed retrospectively the largest mono-institutional series of PTCs with tall-cell features (989 patients) over a 17-year period, re-evaluating tumors based on age at presentation and outcomes in different age groups. We divided patients into three age groups following different criteria (the criterion from the American Joint Committee on Cancer Tumor Node Metastasis (AJCC TNM) guidelines, criterion for the statistical division into tertiles and adolescent/post-adolescent criterion) to analyze the clinicopathological characteristics in different age groups, especially in terms of recurrence-free survival (RFS) and distant recurrence-free survival (DRFS). We obtained three main results: 1. the population is distributed among the different age groups, and therefore, this type of cancer is not exclusively found among those of an older age; 2. in the RFS analysis, we can see a higher probability of local recurrence in the younger and older groups and, unexpectedly, a lower probability of local recurrence in the "median age" group; and 3. in the DRFS analysis, we can observe a higher probability of distant recurrence in older patients. From a molecular perspective, no significant differences in the mutational status of BRAF were detected according to different age groups, while mutations in the TERT promoter were exclusively present in older patients of all age groups, highlighting the potential prognostic implications of TERT promoter mutations in PTCs. In conclusion, the results of this series confirm that TC morphology alone in PTCs does not have the same negative prognostic significance in the younger population as in the older population. The reason for these different outcomes remains unclear and needs further studies.

Immune-related pan-cancer gene expression signatures of patient survival revealed by NanoString-based analyses.

The immune system plays a central role in the onset and progression of cancer. A better understanding of transcriptional changes in immune cell-related genes associated with cancer progression, and their significance in disease prognosis, is therefore needed. NanoString-based targeted gene expression profiling has advantages for deployment in a clinical setting over RNA-seq technologies. We analysed NanoString PanCancer Immune Profiling panel gene expression data encompassing 770 genes, and overall survival data, from multiple previous studies covering 10 different cancer types, including solid and blood malignancies, across 515 patients. This analysis revealed an immune gene signature comprising 39 genes that were upregulated in those patients with shorter overall survival; of these 39 genes, three (MAGEC2, SSX1 and ULBP2) were common to both solid and blood malignancies. Most of the genes identified have previously been reported as relevant in one or more cancer types. Using Cibersort, we investigated immune cell levels within individual cancer types and across groups of cancers, as well as in shorter and longer overall survival groups. Patients with shorter survival had a higher proportion of M2 macrophages and γδ T cells. Patients with longer overall survival had a higher proportion of CD8+ T cells, CD4+ T memory cells, NK cells and, unexpectedly, T regulatory cells. Using a transcriptomics platform with certain advantages for deployment in a clinical setting, our multi-cancer meta-analysis of immune gene expression and overall survival data has identified a specific transcriptional profile associated with poor overall survival.

Autopsy Study of Testicles in COVID-19: Upregulation of Immune-Related Genes and Downregulation of Testis-Specific Genes.

CONTEXT: Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility. OBJECTIVE: Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes. METHODS: Three groups were compared: (a) uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); (b) subjects dying of COVID-19 (virus-negative in testes; n = 15); (c) subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, and immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method. RESULTS: SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups. CONCLUSION: In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

Activation of Type I and Type II Interferon Signaling in SARS-CoV-2-Positive Thyroid Tissue of Patients Dying from COVID-19.

Background: Thyroid dysfunctions have been reported after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. However, the biological mechanisms behind these conditions remain unexplored. Herein, we report on changes of the immune transcriptome in autoptic thyroid tissues of people who have died from coronavirus disease 2019 (COVID-19). Methods: Twenty-five autoptic thyroid specimens of subjects dying from COVID-19 were investigated. Eleven autoptic thyroid specimens of subjects dying from causes other than infectious conditions served as controls. RNA transcripts of 770 immune-related genes together with RNA genomes of multiple coronavirus types were measured by the nCounter system. Reverse transcription-polymerase chain reaction for two SARS-CoV-2 genes was used to assess virus positivity. Results were validated by immunohistochemistry. Results: The SARS-CoV-2 genome and antigens were detected in 9 of 25 (36%) thyroid specimens from the COVID-19 cohort. Virus-negative thyroid tissues from COVID-19 subject did not show changes of gene transcription nor significant numbers of infiltrating immune cells. Conversely, SARS-CoV-2-positive thyroid specimens showed marked upregulation of immune genes, especially those proper of the type I and type II interferon (IFN) pathways. In infected tissues, infiltrates of innate immune cells (macrophages and polymorphonuclear neutrophils) were prevalent. Conclusions: The thyroid gland can be directly infected by the SARS-CoV-2. Infection strongly activates IFN pathways. The direct viral insult combined with an intense immune response may trigger or worsen thyroid conditions in predisposed individuals.

Immune Profiling of Deficient Mismatch Repair Colorectal Cancer Tumor Microenvironment Reveals Different Levels of Immune System Activation.

To understand the immune landscape of deficient mismatch repair colorectal cancer (dMMR CRC) tumor microenvironment, gene expression profiling was performed by the nCounter PanCancer Immune Profiling Panel. This study was conducted retrospectively on 89 dMMR-CRC samples. The expression of CD3, CD8, programmed death-1, and programmed death ligand-1 protein was evaluated on a subset of samples by immunohistochemistry, and lymphocyte density was calculated. A subset of deregulated genes was identified. Functional clustering analysis performed on these genes generated four main factors: antigen processing and presentation, with its major histocompatibility complex-II-related genes; genes correlated with the cytotoxic activity of immune system; T-cell chemotaxis/cell adhesion genes; and T-CD4+ regulator cell-related genes. A deregulation score (DS) was calculated for each sample. On the basis of their DS, tumors were then classified as COLD (DS ≤ -3) to select the samples with a strong down-regulation of the immune system and NOT COLD (DS ≥ -2). The COLD group of patients showed a worse prognosis in terms of survival considering all patients (P = 0.0172) and patients with metastatic disease (P = 0.0031). These results confirm that dMMR-CRCs do not constitute a homogeneous group as concerns the immune system activity of tumor microenvironment. In particular, the distinction between COLD and NOT COLD tumors may improve the management of these two subsets of patients.

Contraception with estradiol valerate and dienogest: adherence to the method.

Purpose: The aim of the study was to examine the discontinuation rate of hormonal contraception with estradiol valerate (E2V) and dienogest (DNG). Patients and methods: We collected data at the Family Planning Clinics of the Departments of Obstetrics and Gynecology of Pisa and Cagliari. We included in the analysis 354 consecutive women using oral contraceptive pills containing E2V and DNG. We analyzed the rate and the reason for discontinuation, classifying the reasons in 5 categories: 1) minor side effects, 2) adverse events, 3) other events not directly caused by the drug or conditions for which the pill could represent a risk factor, 4) no compliance with the method and 5) no further need. Results: Of the 354 women examined, 50.8% had discontinued E2V/DNG pill. Excluding women who stopped the pill because of no further need (10.5%), 27.4% discontinued because of minor side effects, 1.7% discontinued for adverse events, 9.9% because of other events not directly caused by the drug or conditions for which the pill could represent a risk factor and 1.4% because of difficulties with compliance. Irregular bleedings were the main reasons reported for discontinuation. The time to discontinuation for irregular bleedings was significantly (p<0.02) longer in adults than in adolescents and slightly but not significantly longer in women who received information about this possible effect. Conclusion: Unacceptable cycle control was the principal cause of discontinuation of pill with E2V and DNG. An appropriate information about this possible effect may improve adherence to this combined oral contraceptive.

Immune Profiling of Thyroid Carcinomas Suggests the Existence of Two Major Phenotypes: An ATC-Like and a PDTC-Like.

OBJECTIVES: The understanding of the mechanisms underlying thyroid cancer immune escape can lead to the identification of new molecular targets and/or efficacy biomarkers. For this purpose, we performed immune expression profiling in thyroid cancers to obtain a comprehensive view on immune mechanisms activated during cancer progression. METHODS: The study was conducted retrospectively in 25 papillary thyroid carcinomas (PTCs), 14 poorly differentiated thyroid carcinomas (PDTC), 13 anaplastic thyroid carcinomas (ATCs), and 7 normal thyroid (NT) tissue samples. Gene expression profiling was obtained on RNA samples using the Nanostring platform and its nCounter PanCancer Immune Profiling Panel. RESULTS: Gene expression comparison of ATC, PTC, and PDTC vs NT showed high number of regulated genes in cancer samples. In detail, immune-related gene sets were significantly upregulated (ATC > PTC > > PDTC). Most ATC and approximately half of PTC showed a microenvironment infiltrated by macrophages and T-cells with CD8+ effector phenotype, part of which appeared to be functionally exhausted. Conversely, most PDTC, as NT samples, as the remaining part of PTC, displayed a poor or absent infiltration by immune cells. Interestingly, an upregulation of inhibitory immune checkpoint mediators, including PDL1, PDL2, PD1, LAG-3, TIM-3, PVR, and TIGIT, could be detected in ATC and PTC. CONCLUSIONS: These data indicated the existence of two major immune phenotypes in thyroid carcinoma: an ATC-like one, including hot and altered-immunosuppressed tumors and a PDTC-like one, including altered-excluded and cold tumors. Confirmation of the findings in locally advanced or metastatic cancer tissues is expected to have important immunotherapeutic implications.

Association between DNA methylation profile and malignancy in follicular-patterned thyroid neoplasms.

Molecular differentiation between benign (follicular thyroid adenoma, FTA) and malignant (follicular thyroid carcinoma, FTC) thyroid neoplasms is challenging. Here, we explored the genome-wide DNA methylation profile of FTA (n.10) and FTC (n.11) compared to normal thyroid (NT) (n.7) tissues. FTC featured 3,564 differentially-methylated CpGs (DMCpG), most (84%) of them hypermethylated, with respect to normal controls. At the principal component analysis (PCA), the methylation profile of FTA occupied an intermediate position between FTC and normal tissue. A large fraction (n. 2,385) of FTC-associated DMCpG were related (intragenic or within 1500 bp from the transcription start site) to annotated genes (n. 1,786). FTC-hypermethylated genes were enriched for targets of the Polycomb transcriptional repressor complex and the specific histone H3 marks (H3K4me2/me3-H3K27me3) found in chromatin domains known as "bivalent". Transcriptome profiling by RNAseq showed that 7.9% of the DMCpGs-associated genes were differentially expressed in FTC compared to NT, suggesting that altered DNA methylation may contribute to their altered expression. Overall, this study suggests that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to the formation of FTC and that DNA methylation profiling may help differentiating FTCs from their benign counterpart.

Molecular testing in the diagnosis of differentiated thyroid carcinomas.

Different genetic mutations and other molecular alterations in papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) can be detected in fine-needle aspiration (FNA) of thyroid nodules, and can be used successfully to ameliorate cancer diagnosis and management of patients with thyroid nodules. The greatest experience has been obtained with the diagnostic use of BRAF mutation that is strongly specific for malignancy when detected using well-validated techniques. The strongest diagnostic result can be obtained testing FNA samples for a panel of mutations that typically involve TERT, BRAF, PAX8/PPARγ, RAS, and RET/PTC. Finding any of these mutations in a thyroid nodule provides strong indication for malignancy and helps to refine clinical management for a significant proportion of patients with indeterminate cytology. The use of molecular markers, as TERT, BRAF, PAX8/PPARγ, RAS, and RET/PTC, may be considered for patients with indeterminate FNA cytology (FNAC) to help guide management. In patients with indeterminate TIR3 FNA, the combination of precise molecular marker expression analysis with molecular mutations evaluations could ameliorate significantly the accuracy of cancer diagnosis. However other prospective studies are needed to identify more accurate molecular markers. Finally, the knowledge of these molecular pathways has permitted the development of new targeted therapies for aggressive TC.

A six-gene panel to label follicular adenoma, low- and high-risk follicular thyroid carcinoma.

The distinction between follicular thyroid carcinomas (FTCs) and follicular-patterned benign lesions is almost impossible on fine-needle aspiration cytology. Furthermore, minimally invasive FTCs (MI-FTCs) with less than 4 vascular invasion foci generally have an excellent prognosis, but there are exceptions and, so far, no molecular marker appears able to identify them reliably. We aimed to distinguish benign lesions from low- and high-risk FTCs by a small-scale combination of genes. The expression analysis of 75 selected genes was performed on 18 follicular adenomas (FAs), 14 MI-FTCs and 6 widely invasive FTC (WI-FTCs). The mutational status of the RAS genes, TERT promoter and PAX8-PPARG rearrangements was also investigated. Seven samples were mutated, namely 3 MI-FTCs and 4 WI-FTCs. Twenty-five genes were differentially expressed (FDR <0.05) between FAs and WI-FTCs. Six of these (ECM1, RXRG, SDPR, SLC26A4, TIFF3, TIMP1) were also differently expressed among MI-FTCs and FAs or WI-FTCs and were considered to build a classification model, which was tested to classify samples according to their histological class. Hence, 31 out of 38 were correctly classified, and accuracy remained high after cross-validation (27/38). The 2 MI-FTCs incorrectly classified as WI-FTCs harbored both RAS and TERT promoter mutations. The capability of these six genes to stratify benign, low- and high-risk lesions appears to be promising in supporting the diagnosis of indeterminate thyroid nodules.

Analysis of Fusion Genes by NanoString System: A Role in Lung Cytology?

CONTEXT: - Patients with non-small cell lung cancer harboring ALK receptor tyrosine kinase ( ALK), ROS proto-oncogene 1 ( ROS1), and ret proto-oncogene ( RET) gene rearrangements can benefit from specific kinase inhibitors. Detection of fusion genes is critical for determining the best treatment. Assessing rearrangements in non-small cell lung cancer remains challenging, particularly for lung cytology. OBJECTIVE: - To examine the possible application of the multiplex, transcript-based NanoString system (NanoString Technologies, Seattle, Washington) in the evaluation of fusion genes in lung adenocarcinoma samples. DATA SOURCES: - This study is a narrative literature review. Studies about NanoString, gene fusions, and lung adenocarcinoma were collected from PubMed (National Center for Biotechnology Information, Bethesda, Maryland). We found 7 articles about the application of the NanoString system to detect fusion genes on formalin-fixed, paraffin-embedded tumor tissues and one article evaluating the adequacy of lung cytologic specimens for NanoString gene expression analysis. CONCLUSIONS: - To maximize the yield of molecular tests on small lung biopsies, the NanoString nCounter system has been suggested to detect fusion genes. NanoString fusion gene assays have been successfully applied on formalin-fixed, paraffin-embedded tissues. Although there are only a few studies available, the application of NanoString assays may also be feasible in lung cytology. According to available data, the NanoString system could strengthen the routine molecular characterization of lung adenocarcinoma.

Identification of Two Distinct Molecular Subtypes of Non-Invasive Follicular Neoplasm with Papillary-Like Nuclear Features by Digital RNA Counting.

BACKGROUND: The follicular variant (FV) of papillary thyroid cancer (PTC) is one of the most common variants of PTC. Clinically, non-infiltrative FVPTC is considered a low-risk variant of PTC, and the non-invasive encapsulated forms of FVPTC represent a group of thyroid tumors with a particularly good prognosis. Consequently, these neoplasms have been very recently reclassified as non-invasive follicular neoplasms with papillary-like nuclear features (NIFTP). From a molecular standpoint, NIFTP appears to be similar to follicular neoplasms. However, only limited data are currently available regarding their gene expression profile. METHODS: The aim of this study was to identify specific molecular signatures of 26 NIFTPs compared to those of 19 follicular adenomas (FAs) and 18 infiltrative FVPTCs (IFVPTCs). A nanoString custom assay was used to perform mRNA expression analysis. All cases were also genotyped for BRAF, N-, H-, and K-RAS mutations. Samples were grouped on the basis of gene expression profiles by Pearson's correlation and non-negative matrix factorization clustering analysis. Finally, the uncorrelated shrunken centroid machine-learning algorithm was used to classify the samples. RESULTS: The results revealed distinct expression profiles of FAs and IFVPTCs. NIFTP samples can exhibit different expression profiles, more similar to FAs (FA-like) or to IFVPTCs (IFVPTC-like), and these different expression profiles largely depend on the presence of different mutations (RAS or BRAF). CONCLUSION: In conclusion, although further validation of the model is required by using a larger group of prospective cases, these data reinforce the hypothesis that IFVPTC-like NIFTPs might represent precursors of IFVPTC.

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.

Malignant pleural mesothelioma and mesothelial hyperplasia: A new molecular tool for the differential diagnosis.

Malignant pleural mesothelioma (MPM) is a rare asbestos related cancer, aggressive and unresponsive to therapies. Histological examination of pleural lesions is the gold standard of MPM diagnosis, although it is sometimes hard to discriminate the epithelioid type of MPM from benign mesothelial hyperplasia (MH).This work aims to define a new molecular tool for the differential diagnosis of MPM, using the expression profile of 117 genes deregulated in this tumour.The gene expression analysis was performed by nanoString System on tumour tissues from 36 epithelioid MPM and 17 MH patients, and on 14 mesothelial pleural samples analysed in a blind way. Data analysis included raw nanoString data normalization, unsupervised cluster analysis by Pearson correlation, non-parametric Mann Whitney U-test and molecular classification by the Uncorrelated Shrunken Centroid (USC) Algorithm.The Mann-Whitney U-test found 35 genes upregulated and 31 downregulated in MPM. The unsupervised cluster analysis revealed two clusters, one composed only of MPM and one only of MH samples, thus revealing class-specific gene profiles. The Uncorrelated Shrunken Centroid algorithm identified two classifiers, one including 22 genes and the other 40 genes, able to properly classify all the samples as benign or malignant using gene expression data; both classifiers were also able to correctly determine, in a blind analysis, the diagnostic categories of all the 14 unknown samples.In conclusion we delineated a diagnostic tool combining molecular data (gene expression) and computational analysis (USC algorithm), which can be applied in the clinical practice for the differential diagnosis of MPM.

Aberrant expression of anaplastic lymphoma kinase in lung adenocarcinoma: Analysis of circulating free tumor RNA using one-step reverse transcription-polymerase chain reaction.

Lung adenocarcinoma patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements respond well to approved ALK inhibitors. However, to date, limited evidence is available regarding whether using circulating free tumor mRNA to identify aberrant ALK expression is possible, and its feasibility remains to be clearly addressed. The present study evaluated ALK expression by a one-step reverse transcription­polymerase chain reaction (PCR) assay on the circulating free tumor mRNA from 12 lung adenocarcinoma patients. Additionally, the present study tested for ALK rearrangements by fluorescence in situ hybridization (FISH) and immunohistochemistry. A molecular genetic characterization was performed on tumor tissues and plasma samples. Aberrant ALK expression was detected in 2/12 patients using mRNA purified from plasma specimens and the results agreed with the FISH and immunohistochemistry findings of solid biopsy samples. The detection of aberrant ALK expression on circulating free tumor RNA may be feasible using a one­step real­time PCR assay and may be particularly helpful when a solid biopsy sample is not available.

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.