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Table 1: samples included in the RNAseq analysis Figure 1: consort flow diagram Table 2: Comparison of patients' characteristics of the subset of patients included in the RNA-seq analysis and patients recruited in the DECISION study Figure 2: Progression-free survival of the subgroup of patients included in the RNA-seq analysis. Table 3: Pathways_GO_analysis Figure 3: Kaplan-Meyer curves of the three RNA expression groups regarding treatment group in DECISION trial. Figure 4: A,B,C: PFS in papillary, follicular and poorly differentiated histologies, respectively; D,E,F: OS in papillary, follicular and poorly differentiated histologies, respectively Figure 5: Overall survival (OS) analysis based on RNA expression profile from TCGA data of papillary thyroid cancer (Cell, 2014) Figure 6: Analyses based on 121 angiogenesisrelated genes

Several biomarkers have been suggested to have prognostic value in differentiated thyroid carcinomas (DTC) with no validation in the refractory setting, including all tumor subtypes. We aim to correlate RNA expression profiles with survival based on patients included in the DECISION trial. We obtained 247 samples from the 417 patients included in the DECISION study and performed RNAseq analysis (77 million paired-end reads for each sample on HiSeq2000). After quality control, 125 samples were included in the secondary analysis and mapped against the human reference genome (GRCh38) with STAR (v2.5.1b) using ENCODE parameter. Survival analysis was calculated using the Kaplan–Meier method and log-rank test was used for statistical comparison. In this post hoc analysis, we identified three groups of tumors based on their gene expression profile: BRAF-like, RAS-like, and non-BRAF-non-RAS-like (NoBRaL). No significant correlation with sorafenib responders was observed. However, we identified a statistically significant correlation between the RNA-expression profiles and progression-free survival. The BRAF-like profile had a significantly better outcome compared with RAS-like and NoBRaL (11.8, 6.2, and 5.5 months, respectively) [HR: 0.31, 95% confidence interval (CI), 0.17–0.60; P < 0.001 and HR: 0.36 (95% CI, 0.21–0.63); P < 0.001] and HR: 0.36 (95% CI, 0.21–0.63; P < 0.001) and maintained significance as an independent prognostic factor for overall survival in the multivariate analysis for papillary thyroid cancers. To our knowledge, this is the first comprehensive RNA-seq analysis of all histologic subtypes of DTC. The RNA expression profiles identified may suggest a new prognostic parameter to be considered before recommendation of systemic therapies or the design of stratification factors for future clinical trials.

Thyroid nodule (TN) harbouring a thyroid carcinoma are more common in paediatric than adult patients. In paediatric population, the evaluation of a TN should require specific paediatric tools for its diagnostic and therapeutic management. High-resolution ultrasonography and cytological evaluation after fine-needle aspiration biopsy (FNAB) remain the cornerstones of evaluation of TN.To evaluate in paediatric TN for the first time the usefulness and precision of the ultrasound criteria defined by the "Thyroid Imaging Reporting and Data System (EU-TIRADS) 2017 in adults" to establish the ultrasound indication for the practice of FNAB and stratify the risk of malignancy.24 paediatric patients under age 18 years with thyroid nodules were attended in the last 15 years, 24 of them (31 nodules; age: 15.2 ± 2.2 years; 18 women) met the inclusion criteria: FNAB with Bethesda classification and ultrasound with EU-TIRADS score. EU-TIRADS score were evaluated retrospectively. Fourteen patients underwent surgery and the definitive histological diagnosis was obtained, this allowed the calculations of sensitivity, specificity and positive and negative predictive values of the EU-TIRADS and Bethesda classification. Data on the largest diameters of the nodules were collected.Of the overall 31 nodules, the distribution by EU-TIRADS (T) category was: T1 (3.2%), T2: 2 (6.4%), T3: 7 (22.6%), T4: 16 (51.6%) and T5: 5 (16.1%). All malignant nodules were included in EU-TIRADS category 4 or 5. By the other hand, 13 of the 25 benign nodules were also included in the EU-TIRADS 4 category, and one in the 5. The distribution by categories of Bethesda's classification (B): BI: 6 (19.4%), BII: 14 (45.2%), BIII: 5 (16.1%), BIV: 2 (6.5%), BV: 0 and BVI: 4 (12.9%). The pathological diagnosis of the 14 patients who underwent surgery was: 6 papillary carcinomas and 8 with benign lesions: 6 nodular hyperplasia and 2 follicular adenoma. The percentage of malignancy was 42%. The sensitivity of the EU-TIRADS classification to detect malignant nodules was 100%, the specificity was 25%, PPV 44% and NPV 100%. The sensitivity of the Bethesda classification to detect malignant nodules was 86%, the specificity was 75%, PPV 67% and NPV 90%. The analysis of the largest diameter of the nodules did not show statistically significant differences between benign and malignant lesions.EU-TIRADS for ultrasonographic criteria classification in combination with the clinical history is an adequate and reproducible method to estimate suspicion of malignancy of paediatric TN. It is also a reliable diagnostic tool to decide which nodules will be candidates for FNAB.

Resumen Introduccion Los nodulos tiroideos (NT) en la edad pediatrica requieren una evaluacion diagnostica completa porque el riesgo de malignidad es mayor con mas frecuencia que en el adulto. La ecografia es la tecnica de imagen clave para decidir la actitud a seguir. Objetivos Evaluar con caracter inedito la utilidad y precision de los criterios ecograficos definidos por el «Thyroid Imaging Reporting and Data System (EU-TIRADS) 2017» en adultos para estratificar el riesgo de malignidad en pacientes pediatricos con NT y establecer la indicacion de la practica de una puncion aspiracion con aguja fina (PAAF). Pacientes y metodos Se revisaron las historias clinicas de 24 pacientes con NT atendidos en los ultimos 15 anos en un unico centro. Cumplieron los criterios de inclusion establecidos 24 pacientes (31 nodulos; edad: 15,2 ± 2,2 anos; 18 mujeres): PAAF con clasificacion de Bethesda y ecografia con clasificacion EU-TIRADS reevaluadas de forma retrospectiva. A 14 de ellos se les realizo una cirugia y se obtuvo el diagnostico histologico definitivo, esto permitio llevar a cabo los calculos de sensibilidad, especificidad y valores predictivo positivo y negativo de la clasificacion EU-TIRADS y Bethesda. Resultados Del global de 31 nodulos, la distribucion por categoria EU-TIRADS (T) fue: T1 (3,2%), T2: 2 (6,4%), T3: 7 (22,6%), T4: 16 (51,6%) y T5: 5 (16,1%). Todos los nodulos malignos estaban incluidos en la categoria EU-TIRADS 4 o 5. En contraste, 13 de los 25 nodulos benignos fueron integrados tambien en la categoria EU-TIRADS 4, y uno de ellos, en la 5. La distribucion por categorias de la clasificacion de Bethesda (B): B I: 6 (19,4%), BII: 14 (45,2%), BIII: 5 (16,1%), BIV: 2 (6,5%), BV: 0 y BVI: 4 (12,9%). El diagnostico anatomopatologico de los 14 pacientes en los que se realizo la cirugia fue: seis carcinomas papilares y ocho con lesiones benignas; seis hiperplasias nodulares y dos adenomas foliculares. El porcentaje de malignidad fue del 42%. La sensibilidad de la clasificacion EU-TIRADS para detectar nodulos malignos fue del 100%, la especificidad del 25%, el valor predictivo positivo (VPP) del 44% y el valor predictivo negativo (VPN) del 100%. La sensibilidad de la clasificacion Bethesda para identificar nodulos malignos fue del 86%, la especificidad del 75%, VPP 67% y VPN 90%. El analisis del diametro mayor de los nodulos no demostro diferencias estadisticamente significativas entre lesiones benignas y malignas. Conclusiones Los criterios ecograficos en combinacion con la historia clinica representan un metodo adecuado y reproducible para identificar nodulos malignos en la edad pediatrica. La ecografia es una prueba diagnostica fiable para decidir que nodulos seran candidatos a PAAF.

BACKGROUND: Autoimmune thyroid diseases are the most common types of autoimmune diseases, but their physiopathology is still relatively unexplored. Genotype-tissue expression (GTEx) is a publicly available repository containing RNAseq data, including profiles from thyroid. Approximately 14.8% of these glands were affected by focal lymphocytic thyroiditis and 6.3% were annotated as Hashimoto. We interrogated these data to improve the characterization of infiltrating cells and to identify new molecular pathways active in autoimmune thyroiditis. MATERIALS AND METHODS: Histological GTEx images of 336 thyroid samples were classified into three categories, that is, non-infiltrated thyroid, small focal infiltrated thyroid, and extensive lymphoid infiltrated thyroid. Differentially expressed genes among these categories were identified and subjected to in silico pathway enrichment analysis accordingly. CIBERSORTx deconvolution was used to characterize infiltrating cells. RESULTS: As expected, most of the transcriptional changes were dependent on tissue infiltration. Upregulated genes in tissues include—in addition to lineage-specific B and T cell genes—a broad representation of inhibitory immune checkpoint receptors expressed by B and T lymphocytes. CIBERSORTx analysis identified 22 types of infiltrating cells showed that T cells predominate 3:1 over B cells in glands with small infiltrates, only by 1.7:1 in those with large infiltrates. Follicular helper and memory CD4 T cells were significantly more abundant in glands with large infiltrates (p

The risk of recurrence in papillary thyroid carcinoma (PTC) is likely related to the amount of tumour in the metastatic lymph node (LN). Therefore, the current TNM classification (N0/N1) make it necessary to find a method to quantify the LN metastasis (LNM). We propose that the quantitative molecular assay One-Step Nucleic-Acid Amplification (OSNA), which measures the number of cytokeratin-19 (CK-19) mRNA copies as a marker of LNM, could play this role. Our objective was to describe the characteristics of the LNs from PTC, and to compare the morphological characteristics that have been claimed as criteria for metastatic burden with OSNA.Prospective study of LNs from 42 patients. All of the LNs were measured, weighed and analysed by OSNA and also by imprint cytology.A total of 573 LNs were included, 187 (32.6%) of them were OSNA-positives. The global consistency between cytology and OSNA was 87.4%. Significant differences were observed in the CK-19 copy number between the LNMs0.2cm and those3cm, as well as between those from 0.2 to 3cm with respect to those3cm, but not between those0.2cm and those between 0.2 and 3cm. The total tumour load per neck dissection showed no differences based on whether there were ≤5 or5 LNMs.In our series the LNMs3cm show an increased tumour load, but it is unclear if it is necessary to sub-classify the smaller ones as well as the relevance of the number of metastatic nodes according to the cut-off of 5 nodes. We consider that the OSNA analysis avoids the bias of nodal histology and allows for a greater understanding of its real oncological potential.

Purpose To determine the rate of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) in a multi-institutional series from the Iberian Peninsula and describing this NIFTP cohort. Methods Retrospective study of papillary thyroid carcinoma (PTC) or well-differentiated tumours of uncertain malignant potential (WDT-UMP) diagnosed between 2005 and 2015 and measuring >= 5 mm in adult patients from 17 hospitals. Pathological reports were reviewed to determine the cases that fulfil the original criteria of NIFTP and histology was reassessed. Rates were correlated with the number of PTC and its follicular variant (FVPTC) of each institution. Demographic data, histology, management, and follow-up of the reclassified NIFTP cohort were recorded. Results A total of 182 cases with NIFTP criteria were identified: 174/3372 PTC (rate: 5.2%; range: 0-12.1%) and 8/19 WDT-UMP (42.1%). NIFTP rate showed linear correlation with total PTC (p: 0.03) and FVPTC (p: 0.007) identified at each centre. Ultrasound findings were non-suspicious in 60.1%. Fine-needle cytology or core biopsy diagnoses were undetermined in 49.7%. Most patients were treated with total thyroidectomy. No case had nodal disease. Among patients with total thyroidectomy, 89.7% had an excellent response evaluated 1 year after surgery. There were no structural persistence or relapses. Five patients showed residual thyroglobulin after 90 months of mean follow-up. Conclusions NIFTP rate is low but highly variable in neighbouring institutions of the Iberian Peninsula. This study suggests pathologist's interpretation of nuclear alterations as the main cause of these differences. Patients disclosed an excellent outcome, even without using the strictest criteria.

Several biomarkers have been suggested to have prognostic value in differentiated thyroid carcinomas (DTC) with no validation in the refractory setting, including all tumor subtypes. We aim to correlate RNA expression profiles with survival based on patients included in the DECISION trial. We obtained 247 samples from the 417 patients included in the DECISION study and performed RNAseq analysis (77 million paired-end reads for each sample on HiSeq2000). After quality control, 125 samples were included in the secondary analysis and mapped against the human reference genome (GRCh38) with STAR (v2.5.1b) using ENCODE parameter. Survival analysis was calculated using the Kaplan–Meier method and log-rank test was used for statistical comparison. In this post hoc analysis, we identified three groups of tumors based on their gene expression profile: BRAF-like, RAS-like, and non-BRAF-non-RAS-like (NoBRaL). No significant correlation with sorafenib responders was observed. However, we identified a statistically significant correlation between the RNA-expression profiles and progression-free survival. The BRAF-like profile had a significantly better outcome compared with RAS-like and NoBRaL (11.8, 6.2, and 5.5 months, respectively) [HR: 0.31, 95% confidence interval (CI), 0.17–0.60; P < 0.001 and HR: 0.36 (95% CI, 0.21–0.63); P < 0.001] and HR: 0.36 (95% CI, 0.21–0.63; P < 0.001) and maintained significance as an independent prognostic factor for overall survival in the multivariate analysis for papillary thyroid cancers. To our knowledge, this is the first comprehensive RNA-seq analysis of all histologic subtypes of DTC. The RNA expression profiles identified may suggest a new prognostic parameter to be considered before recommendation of systemic therapies or the design of stratification factors for future clinical trials.

We describe a case of a 69-year-old woman with follicular thyroid cancer of long evolution, with thyroidectomy 20 years before, who had remained clinically stable until now, when she started to present abnormal levels of serum thyroglobulin. An 123I whole-body scan showed a high uptake in the upper right half of her abdomen, and an 18F-FDG PET/CT located this focus at the liver's round ligament. Pathology findings after surgery showed the focus to be a differentiated thyroid cancer metastasis. This is an unusual presentation of differentiated thyroid cancer metastasis.

Aldehyde dehydrogenases (ALDHs) are multifunctional enzymes that oxidize diverse endogenous and exogenous aldehydes. We conducted a meta-analysis based on The Cancer Genome Atlas and Gene Expression Omnibus data and detected genetic alterations in ALDH1A1, ALDH1A3, or ALDH3A1, 86% of which were gene amplification or mRNA upregulation, in 31% of nonsmall cell lung cancers (NSCLCs). The expression of these isoenzymes impacted chemoresistance and shortened survival times in patients. We hypothesized that these enzymes provide an oxidative advantage for the persistence of NSCLC. To test this hypothesis, we used genetic and pharmacological approaches with DIMATE, an irreversible inhibitor of ALDH1/3. DIMATE showed cytotoxicity in 73% of NSCLC cell lines tested and demonstrated antitumor activity in orthotopic xenografts via hydroxynonenal-protein adduct accumulation, GSTO1-mediated depletion of glutathione and increased H2O2. Consistent with this result, ALDH1/3 disruption synergized with ROS-inducing agents or glutathione synthesis inhibitors to trigger cell death. In lung cancer xenografts with high to moderate cisplatin resistance, combination treatment with DIMATE promoted strong synergistic responses with tumor regression. These results indicate that NSCLCs with increased expression of ALDH1A1, ALDH1A3, or ALDH3A1 may be targeted by strategies involving inhibitors of these isoenzymes as monotherapy or in combination with chemotherapy to overcome patient-specific drug resistance.

Background Papillary thyroid cancer (PTC) is the most common thyroid carcinoma and exhibits an almost uniformly good prognosis, while anaplastic thyroid cancer (ATC) is less frequent and is one of the most aggressive cancers usually resistant to conventional treatment. Current hypothesis posits that ATC derives from PTC through the progressive acquisition of a discrete number of genomic alterations and implies that the mutational landscape of ATC resembles that of PTC. However, the clinical behaviour of ATC and PTC is radically different. We decided to address the disconnection between the clinical behaviour of ATC and PTC and the proposed model of the progressive development of ATC from PTC. Patients and methods We carried out exome sequencing of DNA from 14 ATC specimens including three cases of concomitant ATC and PTC as well as their corresponding normal DNA from 14 patients. The sequencing results were validated using droplet digital PCR. We carried out immunohistochemistry and immunofluorescence studies of the concomitant ATC and PTC cases. In addition, we integrated our sequencing results with the existing TCGA data. Results Most of the somatic mutations identified in the ATC component differed from the ones in PTC in the cases of concomitant ATC and PTC. The trunks of the phylogenetic trees representing the somatic mutations were short with long branches. In one case of concomitant PTC and ATC specimens, we observed an infiltration of PTC cells within the ATC component. Moreover, we integrated our results with data obtained from TCGA and observed that the most frequent mutations found in ATC presented high cancer cell fraction values and were significantly different from the PTC ones. Conclusion ATC diverge from PTC early in tumour development and both tumour types evolve independently. Our work allows the understanding of the relationship between ATC and PTC facilitating the clinical management of these malignancies.

Introduction Lymphadenectomy is recommended during surgery for papillary thyroid carcinoma when there is evidence of cervical lymph node metastasis (therapeutic) or in high-risk patients (prophylactic) such as those with T3 and T4 tumors of the TNM classification. Selective sentinel lymph node biopsy may improve preoperative diagnosis of nodal metastases. Objective To analyze the results of selective sentinel lymph node biopsy in a group of patients with papillary thyroid carcinoma and no evidence of nodal involvement before surgery. Patients and method A retrospective, single-center study in patients with papillary thyroid carcinoma and no clinical evidence of lymph node involvement who underwent surgery between 2011 and 2013. The sentinel node was identified by scintigraphy. When the sentinel node was positive, the affected compartment was removed, and when sentinel node was negative, central lymph node dissection was performed. Results Forty-three patients, 34 females, with a mean age of 52.3 (±17) years, were enrolled. Forty-six (27%) of the 170 SNs resected from 24 (55.8%) patients were positive for metastasis. In addition, 94 (15.6%) out of the 612 lymph nodes removed in the lymphadenectomies were positive for metastases. Twelve of the 30 (40%) low risk patients (cT1N0 and cT2N0) changed their stage to pN1, whereas 12 of 13 (92%) high risk patients (cT3N0 and cT4N0) changed to pN1 stage. Conclusions Selective sentinel lymph node biopsy changes the stage of more than 50% of patients from cN0 to pN1. This confirms the need for lymph node resection in T3 and T4 tumors, but reveals the presence of lymph node metastases in 40% of T1–T2 tumors.

Resumen Introduccion La linfadenectomia en la cirugia del carcinoma papilar de tiroides se aconseja cuando hay evidencia de metastasis ganglionar cervical (terapeutica) o en pacientes de alto riesgo (profilactica), como en los tumores T3 y T4 de la clasificacion TNM. La tecnica de la biopsia selectiva del ganglio centinela puede mejorar el diagnostico prequirurgico de las metastasis ganglionares. Objetivo Analizar el resultado de la biopsia selectiva del ganglio centinela en un grupo de pacientes con carcinoma papilar de tiroides T sin evidencia de afectacion ganglionar antes de la cirugia. Pacientes y metodo Estudio retrospectivo, unicentrico en el que se incluyeron los pacientes intervenidos entre los anos 2011-2013 que fueran clinicamente N0. La identificacion del ganglio centinela se realizo mediante tecnica isotopica. En todos los casos, se practico linfadenectomia del compartimento afecto si el ganglio centinela era positivo, y del compartimento central en caso de ganglio centinela negativo. Resultados Se incluyeron 43 pacientes, 34 mujeres, con una edad media de 52,3 (±17) anos. De los 170 ganglios centinela resecados, 46 (27%) fueron positivos para metastasis, que correspondian a 24 (55,8%) pacientes. En las linfadenectomias se resecaron 612 ganglios. De ellos, 96 (15,6%) fueron positivos para metastasis. Doce de los treinta (40%) pacientes cT1N0 y cT2N0 pasaron a pN1 tras la biopsia selectiva del ganglio centinela, mientras que 12 de los 13 (92%) pacientes cT3N0 y cT4N0, acabaron siendo pN1. Conclusiones La biopsia selectiva del ganglio centinela recalifica mas del 50% de pacientes de cN0 a pN1. Se confirma la necesidad de vaciamiento ganglionar en los tumores T3 y T4, pero pone al descubierto la presencia de metastasis linfaticas en el 40% de los T1-T2.

Objective Lymphadenectomy in papillary thyroid carcinoma (PTC) is controversial. It is indicated whenever metastases have been proven before or during surgery and as a prophylactic treatment in high-risk patients. However, 30–50% of cN0 patients become pN1 postoperatively. In PTC, selective-sentinel-lymph-node-biopsy (SLNB) with conventional intraoperative analysis is 8% false negative. One-step nucleic acid amplification (OSNA) is a molecular technique which allows real-time detection of mRNA encoding for cytokeratin 19. OSNA has been introduced in intraoperative analysis of several tumors to reduce false-negative rates and distinguish micrometastasis from macrometastasis. Our objective was to evaluate the impact of the introduction of OSNA in the intraoperative evaluation of the sentinel node (SN) in PTC. Design We analyzed a series of 35 patients subjected to SLNB. Methods All the dissected nodes, SN and non-SN, were evaluated with OSNA and cytology. Results We obtained a total of 110 SN. SLNB proved positive in 14 patients (40%) with cytology and in 23 (65.7%) with OSNA (P P = 0002). The majority of patients with micrometastasis in SN showed only micrometastasis in lymphadenectomy. Conclusions The present study shows selective-sentinel-lymph-node-biopsy with one-step nucleic acid amplification technique to be feasible in papillary thyroid carcinoma. The quantitative nature of one-step nucleic acid amplification paves the way toward a more personalized surgical approach, limiting lymphadenectomy to patients with intraoperative evidence of macrometastasis in the sentinel node.

Background Role of Tg levels as a biomarker in RAI-R DTC during MKI is unclear. Falling levels of Tg after starting MKI treatment has been previously reported, with a higher reduction in patients who achieve response. However, transient Tg oscillations are a common phenomenon during therapy without a clear correlation with progressive disease (PD). In the present study, we analyze the correlation between Tg oscillations and progression-free survival (PFS) in patients with RAI-R DTC during MKI treatment. Methods Thirty-seven consecutive patients were reviewed. Inclusion criteria were: pathology confirmed RAI-R DTC, age >18 years, MKI treatment in the first or second line and serial Tg determinations along with the follow-up. Variations in Tg levels during MKI treatment were evaluated. As exploratory hypothesis, we considered the “Tg elevation non-PD related”, defined as a significant elevation of Tg higher than 50% compared with previous measure, not related with PD by RECIST criteria, followed by a Tg reduction in a subsequent determination, in patients with an initial Tg reduction after starting MKI treatment. Results 33 patients were included in the final analysis. Most MKI treatments were Sorafenib (44%), Lenvatinib (27%), Vandetanib (10%) and Axitinib (8%). Median duration of treatment was 15.5 months, and median PFS 20.2 months. Patients with any Tg levels reduction compared with baseline along MKI treatment showed a better PFS (23.5 vs. 3.6 months, p 50% from baseline on the first month after starting MKI also had a benefit in PFS (27.7 vs. 14.2, p 0.02). 20.6% of cases had the nadir of Tg response beyond the first month of treatment. 34.4% of cases presented Tg elevation non-PD related along with follow-up (median number of elevations per patient: 2), with a better PFS compared with patients with other Tg oscillation patterns (29.9 vs. 15.5 months, p0.008). Conclusions Tg elevation non-PD related was associated with a better PFS compared with patients with other Tg variation patterns. Treatment with MKIs should not be stopped or changed based on Tg variations. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure J. Hernando Cubero: Speaker Bureau / Expert testimony: Eisai, Ipsen, Roche, Angelini Pharma; Travel / Accommodation / Expenses: Ipsen, Novartis, AAA, Roche, AstraZeneca, Eisai. All other authors have declared no conflicts of interest.

Purpouse: One Step Nucleic Acid Amplification (OSNA) has been previously proposed for the diagnosis of lymph node metastases (LNMs) from several malignant conditions by quantifying the number of copies of cytokeratin 19 mRNA. Our aim was to evaluate the results obtained by OSNA in the lymph nodes of patients with papillary thyroid carcinoma (PTC) by comparing our results with the findings observed using standard pathological examination.Fifty human lymph nodes (from five patients with diagnosed PTC) were studied. Each node was divided into two: one half was used for molecular study ("OSNA-node"), and the other half was used for conventional staining with hematoxylin and eosin ("HE-non-OSNA node"). Three cytological imprints using Papanicolaou and May-Grunwald-Giemsa strains were obtained from both node halves. The results from each technique were compared, and ROC analysis was performed.The OSNA study showed 22 positive samples for LNM (44%), which demonstrate a high concordance rate with the results observed using conventional pathological examination (cytology of "OSNA-node" and HE of "Non-OSNA node") with specificity and sensitivity values greater than 86% and 89%, respectively. However, both comparisons differed in the number of copies of mRNA as the best cut-off (260 copies in the first case and 93 in the second case).The OSNA results for the detection of LNM in patients with PTC are comparable with those observed using conventional techniques. However, its quantitative nature could be useful to more accurately detect lymph node involvement.

Context: Global DNA hypomethylation is a major event for the development and progression of cancer, although the significance in thyroid cancer remains unclear. Therefore, we aimed to investigate its role in thyroid cancer progression and its potential as a prognostic marker.Methods: Global hypomethylation of Alu repeats was used as a surrogate marker for DNA global hypomethylation, and was assessed using the Quantification of Unmethylated Alu technique. Mutations in BRAF and RAS were determined by Sanger sequencing.Results: Ninety primary thyroid tumors were included [28 low-risk differentiated thyroid cancer (DTC), 13 pediatric DTC, 33 distant metastatic DTC, 7 poorly differentiated thyroid cancer (PDTC), and 9 anaplastic thyroid cancer (ATC)], as well as 24 distant metastases and 20 normal thyroid tissues. An increasing hypomethylation was found for distant metastatic DTC [median, 4.0; interquartile range (IQR), 3.1 to 6.2] and PDTC/ATC (median, 9.3; IQR, 7.0 to 12.1) as compared with normal thyroid tissue (median, 2.75; IQR, 2.30 to 3.15), whereas low-risk and pediatric DTC were not affected by hypomethylation. Alu hypomethylation was similar between distant metastases and matched primary tumors. Within distant metastatic DTC, Alu hypomethylation was increased in BRAF vs RAS mutated tumors. Kaplan-Meier and Cox regression analyses showed that thyroid cancer-related and all-cause mortality were associated with tumor hypomethylation, but this association was lost after adjustment for thyroid cancer risk category.Conclusion: Distant metastatic DTC, PDTC, and ATC were increasingly affected by global Alu hypomethylation, suggesting that this epigenetic entity may be involved in thyroid cancer progression and dedifferentiation.

Lymphadenectomy is recommended during surgery for papillary thyroid carcinoma when there is evidence of cervical lymph node metastasis (therapeutic) or in high-risk patients (prophylactic) such as those with T3 and T4 tumors of the TNM classification. Selective sentinel lymph node biopsy may improve preoperative diagnosis of nodal metastases.To analyze the results of selective sentinel lymph node biopsy in a group of patients with papillary thyroid carcinoma and no evidence of nodal involvement before surgery.A retrospective, single-center study in patients with papillary thyroid carcinoma and no clinical evidence of lymph node involvement who underwent surgery between 2011 and 2013. The sentinel node was identified by scintigraphy. When the sentinel node was positive, the affected compartment was removed, and when sentinel node was negative, central lymph node dissection was performed.Forty-three patients, 34 females, with a mean age of 52.3 (±17) years, were enrolled. Forty-six (27%) of the 170 SNs resected from 24 (55.8%) patients were positive for metastasis. In addition, 94 (15.6%) out of the 612 lymph nodes removed in the lymphadenectomies were positive for metastases. Twelve of the 30 (40%) low risk patients (cT1N0 and cT2N0) changed their stage to pN1, whereas 12 of 13 (92%) high risk patients (cT3N0 and cT4N0) changed to pN1 stage.Selective sentinel lymph node biopsy changes the stage of more than 50% of patients from cN0 to pN1. This confirms the need for lymph node resection in T3 and T4 tumors, but reveals the presence of lymph node metastases in 40% of T1-T2 tumors.

Background: Current methods based on fine-needle aspiration biopsy (FNAB) are not sufficient to distinguish among follicular thyroid lesions, follicular adenoma (FA), follicular thyroid carcinoma (FTC), and the follicular variant of papillary thyroid cancer (FVPTC). Furthermore, none of the immunohistochemical markers currently available are sensitive or specific enough to be used in the clinical setting, necessitating a diagnostic hemithyroidectomy. The aim of this study was to identify proteins of value for differential diagnosis between benign and malignant thyroid follicular lesions. Methods: This retrospective analysis is based on an assessment of the immunoexpression of 19 proteins on 81 benign thyroid lesions (FA) and 50 malignant tumors (FTC/FVPTC). The resulting expression profile allowed the design of a scoring system model to improve the differential diagnosis of benign and malignant thyroid lesions. The model was validated using an independent series of 69 FA and 40 FTC and an external series of 40 nodular hyperplasias, and was further tested in a series of 38 FNAB cell blocks. Results: A model based on the nuclear and cytoplasmic expression of APLP2, RRM2, and PRC1 discriminated between benign and malignant lesions with 100% sensitivity in both main and validation groups, with specificities of 71.3% and 50.7%, respectively. For the nodular hyperplasia series, specificity reached 94.8%. Finally, in FNAB samples, the sensitivity was 100% and the specificity was 45% for discrimination between benign and malignant lesions. Conclusions: These findings suggest that the identified APLP2, RRM2, and PRC1 signature could be useful for distinguishing between benign (FA) and malignant (FTC and FVPTC) tumors of the thyroid follicular epithelium.

Autoimmune thyroid diseases (AITDs), i.e., Graves’ disease (GD) and Hashimoto thyroiditis (HT), are the most prevalent organ-specific autoimmune diseases, but their pathogenesis is still incompletely understood. The PD-1/PD-L1 pathway is an important mechanism of peripheral tolerance that has not been investigated in AITDs. Here, we report the analysis of the expression of PD-1, PD-L1 and PD-L2 in PBMCs, infiltrating thyroid lymphocytes (ITLs) and in thyroid follicular cells (TFCs) in GD, HT and multinodular goiter (MNG) patients and healthy controls PBMCs (HC). By combining flow cytometry, tissue immunofluorescence and induction experiments on primary and thyroid cell line cultures, we show that: 1) while PD-1+ T cells are moderately expanded in PBMCs from GD vs HC, approximately half of T cells in the infiltrate are PD-1+ including some PD-1hi; 2) PD-L1, but not PD-L2, is expressed by 81% of GD glands and in 25% of non-autoimmune glands; 3) PD-L1, was expressed by TFCs in areas that also contain abundant PD-1 positive T cells but; 4) co-localization in TFCs indicated only partial overlap between the smaller areas of the PD-L1+ and the larger areas of HLA class II+ expression; 5) IFNγ is capable of inducing PD-L1 in >90% of TFCs in primary cultures and cell lines. Collectively these results indicate that the PD-1/PD-L1 axis is operative in AITD glands and may restrain the autoimmune response. Yet the discrepancy between easy induction in vitro and the limited expression in vivo (compared to HLA) suggests that PD-L1 expression in vivo is partially inhibited in GD and HT glands. In conclusions 1) the PD-1/PD-L1 pathway is activated in AITD glands but probably not to the extent to inhibit disease progression and 2) Thyroid autoimmunity arising after PD-1/PD-L1 blocking therapies in cancer patients may result from interfering PD-1/PD-L1 tolerance mechanism in thyroid with minimal (focal) thyroiditis. Finally acting on the PD-1/PD-L1 pathway could be a new approach to treat AITD and other organ-specific autoimmunity in the future.

Lymphadenectomy in papillary thyroid carcinoma (PTC) continues to be controversial. A better staging method is needed to provide adequate individual surgical treatment. SPECT/CT lymphoscintigraphy and sentinel lymph node (SLN) biopsy may improve lymphatic staging and surgical treatment. Our main objectives were to describe the lymphatic drainage of PTC using lymphoscintigraphy, to evaluate the lymphatic spread (comparing SLN and lymphadenectomy results) and to analyse the impact of SLN identification in surgery. We prospectively studied 24 consecutive patients with PTC (19 women; mean age 52.7 years, range 22–81 years). The day before surgery, lymphoscintigraphy with ultrasound-guided intratumoral injection (99mTc-nanocolloid, 148 MBq) was performed, obtaining planar and SPECT/CT images. All patients underwent total thyroidectomy, SLN biopsy (hand-held gamma probe) with perioperative analysis, central compartment node dissection, or laterocervical lymphadenectomy if perioperative stage N1b or positive SLNs in this lymphatic basin. Lymphoscintigraphy revealed at least one SLN in 19 of 24 patients (79 %) on planar and SPECT/CT images, and in 23 of 24 patients (96 %) during surgery using a hand-held gamma probe. Lymph node metastases were detected with classical perioperative techniques (ultrasound guidance and surgical inspection) in 3 of 24 patients, by perioperative SLN analysis in 10 of 23, and by definitive histology in 13 of 24. The false-negative (FN) ratio for SLN was 7.7 % (one patient with bulky lymph nodes). The FN ratio for perioperative frozen sections was 15.4 % (two patients, one with micrometastases, the other with bilateral SLN). Lymphatic drainage was only to the central compartment in 6 of 24 patients (3 of the 6 with positive SLNs for metastases), only to the laterocervical basin in 5 of 24 patients (all unilateral, 2 of 5 positive SLNs) and to the central and laterocervical compartments in 12 of 24 patients (6 of 12 and 3 of 12 positive SLNs, respectively). Lymphoscintigraphy reveals the lymph node drainage in a high proportion of patients. It detects laterocervical drainage in a significant percentage of patients, allowing the detection of occult lymph node metastases and improving the surgical management in PTC.

The correct cytological discrimination between benign and malignant lesions is the first invaluable point for the adequate clinical and/or surgical management of head and neck lesions. This chapter offers a detailed and specific overview of both the common and rare benign and malignant lesions of the head and neck district. We deem the increasing role of cytology in some areas, such as nasal, sinonasal, oropharyngeal, major and minor salivary glands, and odontogenic, offering specific diagnostic features in a territory that has been mainly and largely diagnosed from a histological perspective. A specific section underlines the cytological features of head and neck cystic lesions, and a focused attention is directed to the cytological evaluation of benign and malignant mesenchymal and lymphoid lesions. Furthermore, the growing number of different cytological methods (i.e., conventional cytology, liquid-based cytology, and cell blocks) is highlighted mainly because of the application of ancillary techniques such as immunocytochemistry, flow cytometry and molecular analysis. A final detailed section points to all the possible and different benign and malignant thyroid lesions which are assessed with both conventional and liquid-based cytology. The conclusive point is a discussion about the problem of reporting thyroid diagnoses as emerged in the different thyroid classification systems used. All of these critical limits are analyzed in detail in the present chapter with appropriate references and pertinent illustrations. In this regard and taken together both all the advantages and the pitfalls and drawbacks, cytology might be the first and worldwide diagnostic tool in achieving a correct diagnosis and guiding the most appropriate management.

Resumen Para un patologo preparado, reconocer un tumor cuando presenta la morfologia caracteristica y esta en su localizacion habitual es facil. Pero cuando se presenta en un lugar insolito y ademas la biopsia para diagnostico es pequena, es facil caer en un error de orientacion y, aunque a veces las tecnicas auxiliares nos puedan ayudar, en algunas ocasiones no se concreta el diagnostico hasta que una biopsia completa o la extirpacion del tumor nos permiten ver la totalidad de la lesion, con la sobrecarga de tiempo, riesgo para el paciente y gasto economico que ello conlleva. El Prof. Rosai describe esta situacion con la historia que contaba Lauren V. Ackerman y que titulo «El hombre de Estambul»: el empecinamiento en no reconocer un tumor porque no esta en «su» lugar. Aportamos dos casos de sarcomas de partes blandas que se presentaron en localizaciones que podriamos llamar «invertidas»: un sarcoma sinovial mandibular y un mioepitelioma maligno yuxtaarticular en un dedo del pie y exponemos las dificultades que presentaron para su diagnostico inicial.

In an attempt to identify molecules that clearly reflect the oncogenic role of cell signaling pathways in human tumors, we propose a concept we term “funnel factor”, a factor where several oncogenic signals converge and drive the proliferative signal downstream. In studies done in various tumor types, the expression of key cell signaling factors, including Her1 and Her2 growth factor receptors, as well as the RAS-RAF-mitogen-activated protein kinase and the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathways was correlated with the associated clinicopathologic characteristics of these tumors. The downstream factors p70, S6, 4E-binding protein 1 (4E-BP1), and eukaryotic translation initiation factor 4E, which play a critical role in the control of protein synthesis, survival, and cell growth, were also analyzed. We found that phosphorylated 4E-BP1 (p-4E-BP1) expression in breast, ovary, and prostate tumors is associated with malignant progression and an adverse prognosis regardless of the upstream oncogenic alterations. Thus, p-4E-BP1 seems to act as a funnel factor for an essential oncogenic capability of tumor cells, self-sufficiency in growth signals, and could be a highly relevant molecular marker of malignant potential. Further investigation into this concept may identify additional funnel factors in the oncogenic pathways and provide potential therapeutic targets. [Cancer Res 2007;67(16):7551–5]

Helical computerized tomography (CT) and magnetic resonance imaging (MRI) are used for staging of hepatocellular carcinoma (HCC) prior to curative treatments but underestimate tumor extension in 30% to 50% of cases when compared with pathologic explants. This study compares a new technology, MRI angiography (MRA), with triphasic helical CT in detection of HCC. Fifty cirrhotic patients, 29 with HCC, undergoing liver transplantation were analyzed. MRA was performed with a 3-D breath-hold fast spoiled gradient-echo sequence by using an effective section thickness of 2 to 2.5 mm. The gold standard was the pathologic examination (liver cut into 5-mm slices). One hundred twenty-seven lesions were identified at the explant: 76 HCC, 13 high-grade dysplastic nodules, 31 macroregenerative nodules, 7 hemangiomas. Diameter of the main HCC nodules was 29 +/- 14 mm and 11 +/- 7 mm for the 47 additional nodules. On a per nodule basis, sensitivity of MRA was superior to CT (58/76 [76%] vs. 43/70 [61%], respectively, P =.001). Sensitivity of MRA for detection of additional nodules decreased with size (20 mm: 6/6 [100%]; 10-20 mm: 16/19 [84%];10 mm: 7/22 [32%]) and was superior to CT for nodules 10 to 20 mm (84% vs. 47%, P =.016). Nonspecific hypervascular nodules5 mm at MRA were HCC in two thirds of the cases. In conclusion, MRA has a high diagnostic accuracy for HCCor =10 mm and is more sensitive than triphasic helical CT in nodules sized 10 to 20 mm. MRA is the optimal technique for HCC staging prior to curative therapies.

6083 Background: In DECISION, sorafenib significantly impacted progression-free survival (PFS) and response rate (RR) in radioactive-iodine refractory DTC. The aim of this biomarker study was to identify RNA expression profiles related with PFS, overall survival (OS) and RR and to describe the expression profiles of DTC histologies. Methods: Of the 417 patients in the trial, 247 had sufficient formalin fixed paraffin embedded archival tumor material for RNAseq. We generated on average 77 million paired-end reads for each sample on HiSeq2000 (Illumina). RNAseq reads were mapped against the human reference genome (GRCh38) with STAR (v2.5.1b) using ENCODE parameters. 125 samples had sufficient quality to be included in the analysis. Results: The analysis subset included 68 sorafenib and 57 placebo patients (PFS 10.3 vs 7.4 months, HR: 0.62 CI 95% 0.38-0.99, p = 0.046). Unsupervised clustering using the 100 most variable genes identified 3 groups: BRAF-like (included most of the BRAF-mutated tumors), RAS-like (included most of the RAS mutated tumors) and non-BRAF-non-RAS-like group (included most wild-type tumors). These groups, based on the mutational profile, can be correlated with tumor type: the papillary BRAF-mutant, the follicular wild-type, and a third group with papillary, follicular and poorly differentiated with predominant RAS mutations. A Student t-test comparing papillary and follicular histologies revealed a signature of 283 genes with significantly different expression that, within the papillary tumors, identifies a subset with an expression profile more similar to follicular. No RNA signatures correlating with benefit from sorafenib were identified. Conclusions: While papillary and follicular thyroid cancers have significantly different RNA expression profiles, a subset of papillary has been identified with an expression profile more similar to follicular. In addition, a unified RAS-like expression profile spans subsets of papillary, follicular, and poorly differentiated thyroid cancers, suggesting that tumor biology can be similar across histologies. Clinical trial information: NCT00984282.

Purpose: To analyze the expression of PTOV1 in high-grade prostatic intraepithelial neoplasia (HG-PIN) and to explore its usefulness to predict prostate cancer in patients with isolated HG-PIN in needle biopsy (prostate needle biopsy). Experimental Design: PTOV1 expression in HG-PIN lesions from 140 patients was analyzed by immunohistochemistry in a semiquantitative manner (Histo-score). HG-PIN derived from 79 radical prostatectomies for prostate cancer and from 11 cistoprostatectomies for bladder cancer without prostate cancer were used as positive and negative controls, respectively. Fifty patients with HG-PIN without concomitant cancer at their first prostate needle biopsy were chosen as the study group. Patients were followed by a mean of 2.5 repeated prostate needle biopsies (1-5), during a mean period of 12.4 months (1-39). Results: PTOV1 expression in HG-PIN from radical prostatectomies showed a significantly higher Histo-score (162.6) compared with specimens from cistoprostatectomies (67.0). In the study group, PTOV1 expression was significantly higher in samples with cancer in the follow-up (11 patients, 22%) compared with samples in which cancer was not detected (151.4 versus 94.6). PTOV1 expression was the only independent predictor of cancer in the multivariate analysis and the area under the curve was 0.803 (95% confidence interval, 0.728-0.878). A threshold of 100 for PTOV1 expression provided 90.9% sensitivity, 51.3% specificity, 34.5% positive predictive value, and 95.2% negative predictive value. Conclusions: PTOV1 is overexpressed in HG-PIN associated with cancer and is a potential marker for studying the carcinogenesis and progression of prostate cancer. Prostate needle biopsy with PTOV1 expression in HG-PIN above a threshold of 100 should be repeated immediately for the likely presence of undiagnosed cancer.

6033 Background: Anaplastic thyroid cancer (ATC) is a rare and highly lethal malignancy. Chemotherapeutic agents and surgery have had no impact on local control or prognosis and novel actionable th...

9524 Background: Activation of the PI3-K/Akt/mTOR signal transduction pathway contributes to the development and progression of tumors by prevention of apoptosis and deregulation of the cell cycle. Dissecting the molecular events of this pathway may provide instrumental knowledge with implications for the development of PI3K/Akt/mTOR inhibitors and multi-signalling inhibitory strategies. Methods: We have analysed 121 paraffin-embedded human malignant tumors: 25 gastric, 25 colon, 23 breast, 19 prostate, 19 glioblastoma and 10 pancreas, with a complete immunohistochemistry profile including multiple phosphorylated (p) downstream proteins: pAkt, p4EBP1, pp70S6K and pS6K. The levels of expression were evaluated as percentage and intensity of stained cells (Hscore). Results: Globally, there was a significant correlation between the levels of pAkt, p4EBP1, pp70S6K and pS6K (Spearman's test, p

Tumor resection in papillary thyroid carcinoma (PTC) is often accompanied by lymph node (LN) removal of the central and lateral cervical compartments. One-step nucleic acid amplification (OSNA) is a polymerase chain reaction-based technique that quantifies cytokeratin 19 (CK19) messenger RNA copies. Our aim is to assess the value of OSNA in detection of LN metastases in PTC, in comparison with imprints and microscopic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue. A total of 387 LNs from 37 patients were studied. From each half LN, 2 imprints were taken and analyzed with hematoxylin and eosin (HE) and CK19 immunostaining. One half of the LN was submitted to OSNA and one half to FFPE processing and HE and CK19 staining. For concordance analysis, every single LN was considered as a case. A group of 11 cases with discordant results between OSNA and HE/CK19 FFPE sections were subjected to additional FFPE serial sectioning and HE and CK19 staining. We found a high degree of concordance between the assays used, with sensitivities ranging from 0.81 to 0.95, and specificities ranging from 0.87 and 0.98. OSNA allowed upstaging of patients from pN0 to pN1, in comparison with standard pathologic analysis. Identification of a metastatic LN with more than 15000 CK19 messenger RNA copies predicted the presence of a second LN with macrometastasis (5000 copies). In summary, the study shows that OSNA application in sentinel or suspicious LN may be helpful in assessing nodal status in PTC patients.