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Molecular testing (MT) of thyroid fine-needle aspiration (FNA)-derived genetic material is commonly used to assess malignancy risk for indeterminate cases. The Bethesda System for Reporting Thyroid Cytopathology (TBS) provides limited guidance for the appropriate use of category III (atypia of undetermined significance [AUS]). The authors combined MT with cytomorphology to monitor AUS diagnoses in a cytopathology laboratory.Neoplasia-associated genetic alterations (NGAs) were determined by MT of preoperative FNA biopsies or resected malignancies and were categorized as BRAF V600E mutations, RAS-like mutations (HRAS, NRAS, or KRAS mutations or non-V600E BRAF mutations), or other mutations.Among 7382 thyroid FNA biopsies, the AUS rate was 9.3% overall and ranged from 4.3% to 24.2% among 6 cytopathologists (CPs) who evaluated150 cases. The ratio of specimens falling into TBS category III to specimens falling into category VI (malignant) (the III:VI ratio) was 2.4 overall (range, 1.1-8.1), and the ratio of specimens falling into TBS categories III and IV (follicular neoplasm or suspicious for follicular neoplasm) combined (III+IV) to specimens falling into category VI (the [III+IV]:VI ratio) was 2.9 overall (range, 1.4-9.5). MT was performed on 588 cases from 560 patients (79% women) with a median age of 56 years (range, 8-89 years). BRAF V600E mutation was the most common (76% of cases) in TBS category VI and was rare (3%) in category III. RAS-like mutations were most common in TBS categories III (13%), IV (25%), and V (suspicious for malignancy) (17.5%). The NGA rate in AUS cases fell between 5% and 20% for 5 of 6 CPs and did not correlate with the III:VI ratio or the (III+IV):VI ratio.Lack of correlation between the NGA rate and easily calculable diagnostic ratios enables the calibration of diagnostic thresholds, even for CPs who have normal metrics. Specifically, calculation of the NGA rate and the III:VI ratio may allow individual CPs to determine whether they are overcalling or undercalling cases that other CPs might otherwise recategorize.

Loss of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) expression by CpG promoter hypermethylation is associated with metastasis in gastroenteropancreatic neuroendocrine tumors; however, the mechanism of how UCHL1 loss contributes to metastatic potential remains unclear. In this study, we first confirmed that the loss of UCHL1 expression on immunohistochemistry was significantly associated with metastatic tumors in a translational pancreatic neuroendocrine tumor (PNET) cohort, with a sensitivity and specificity of 78% and 89%, respectively. To study the mechanism driving this aggressive phenotype, BON and QGP-1 metastatic PNET cell lines, which do not produce UCHL1, were stably transfected to re-express UCHL1. In vitro assays, RNA sequencing and reverse phase protein array (RPPA) analyses were performed comparing empty-vector negative controls and UCHL1-expressing cell lines. UCHL1 re-expression is associated with lower anchorage-independent colony growth in BON cells, lower colony formation in QGP cells and a higher percentage of cells in the G0/G1 cell-cycle phase in BON and QGP cells. On RPPA proteomic analysis, there was an upregulation of cell-cycle regulatory proteins CHK2 (1.2-fold change, P = 0.004) and P21 (1.2-fold change, P = 0.023) in BON cells expressing UCHL1; western blot confirmed upregulation of phosphorylated CHK2 and P21. There were no transcriptomic differences detected on RNA sequencing between empty-vector negative controls and UCHL1-expressing cell lines. In conclusion, UCHL1 loss correlates with metastatic potential in PNETs and its re-expression induces a less aggressive phenotype in vitro, in part by inducing cell-cycle arrest through posttranslational regulation of phosphorylated CHK2. UCHL1 expression should be considered as a functional biomarker in detecting PNETs capable of metastasis.

Background and objectives Hurthle cell carcinoma (HCC) is an unusual and relatively rare type of differentiated thyroid cancer. Currently, cytologic analysis of fine-needle aspiration biopsy is limited in distinguishing benign Hurthle cell neoplasms from malignant ones. The aim of this study was to determine whether differences in the expression of specific genes could differentiate HCC from benign Hurthle cell nodules by evaluating differential gene expression in Hurthle cell disease. Methods Eighteen benign Hurthle cell nodules and seven HCC samples were analyzed by whole-transcriptome sequencing. Bioinformatics analysis was carried out to identify candidate differentially expressed genes. Expression of these candidate genes was re-examined by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was quantified by immunohistochemistry. Results Close homolog of L1 (CHL1) was identified as overexpressed in HCC. CHL1 was found to have greater than 15-fold higher expression in fragments per kilobase million in HCC compared with benign Hurthle cell tumors. This was confirmed by qRT-PCR. Moreover, the immunoreactivity score of the CHL1 protein was significantly higher in HCC compared with benign Hurthle cell nodules. Conclusions CHL1 expression may represent a novel and useful prognostic biomarker to distinguish HCC from benign Hurthle cell disease.

Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs). Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clinically annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA. TMA-1 (n = 80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n = 51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA-2, 20 (74%) of the ACC tumors demonstrated PTTG1 nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p

Metastatic papillary thyroid carcinoma without an identifiable primary tumor despite extensive microscopic examination of the thyroid gland is a rare but true phenomenon. We retrieved seven of such cases and described in details the clinical and pathologic features of these tumors. BRAF V600E immunohistochemistry and Sequenom molecular profile were conducted in selected cases. All patients harbored metastatic disease in the central (n = 3), lateral (n = 3), or both neck compartments (n = 1). The histotype of the metastatic disease was papillary thyroid carcinoma (PTC, n = 5), poorly differentiated thyroid carcinoma (PDTC) in association with a PTC columnar variant (n = 1), and anaplastic thyroid carcinoma (ATC) in association with a PTC tall cell variant (n = 1). Fibrosis was present in the thyroid of five patients. All patients with PTC were alive without evidence of recurrence. The 76-year-old patient with PDTC did not recur and died of unknown causes. Finally, the patient with ATC was alive with distant metastasis at last follow up. The median follow up for this cohort was 2.2 years (range 0.8 – 17). BRAF V600E was detected in 4 of 6 cases by immunohistochemistry. In conclusion, metastatic nodal disease without identifiable thyroid primary is a rare but real phenomenon of unknown mechanisms. Although most tumors are low grade and well differentiated, aggressive behavior due to poorly differentiated or anaplastic carcinoma can happen. Most cases are BRAF V600E positive thyroid tumors. A papillary carcinoma phenotype is found in all reported cases.

Background Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. Methods Thyroid fine-needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. Results Fifty-six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7-77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0-23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0-42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell-free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false-negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. Conclusions Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.

Background The tall cell variant of papillary thyroid carcinoma (PTC-TC) has been associated with aggressive features including extrathyroidal extension, higher rate of lymph node and distant metastases, and higher recurrence rate. We aimed to evaluate the cytomorphologic features of PTC-TC on ThinPrep (TP) along with its diagnostic efficacy to detect PTC-TC. Methods Preoperative cytology samples from 30 cases of histologically-proven PTC-TC and 30 classical PTC controls were selected for this study. TP preparations were evaluated for varying architectural and cytomorphologic features. Results Tall cells were present in the majority of PTC-TC cases and were located at the periphery of cell clusters and as single cells. Cytoplasmic cuff along the periphery of cell clusters and soap-bubble pseudoinclusions were very specific features of PTC-TC, when present. PTC-TC cases were more likely to show abundant oncocytic cytoplasm and distinct cell borders. Cytoplasmic tails were more likely to be present and more numerous in PTC-TC. The presence of nuclear grooves, papillary architecture, and giant cells were not reliable distinguishing features of PTC-TC vs controls. Conclusion Our results indicate that tall cell cytomorphologic and architectural features in PTC are identifiable on TP.

Adrenocortical carcinoma (ACC) is a rare tumor type with a poor prognosis and few therapeutic options.Assess prostate-specific membrane antigen (PSMA) expression as a potential novel therapeutic target for ACC.Expression of PSMA was evaluated in benign and malignant adrenal tumors and 1 patient with metastatic ACC.This study took place at a tertiary referral center.Fifty adrenal samples were evaluated, including 16 normal adrenal glands, 16 adrenocortical adenomas, 15 primary ACC, and 3 ACC metastases.Demographics, PSMA expression levels via real-time quantitative polymerase chain reaction and immunohistochemistry and whole-body positron emission tomography-computed tomography standardized uptake values for 1 patient.qPCR demonstrated an elevated level of PSMA in ACC relative to all benign tissues (P.05). Immunohistochemistry localized PSMA expression to the neovasculature of ACC and confirmed overexpression of PSMA in ACC relative to benign tissues both in intensity and percentage of vessels stained (78% of ACC, 0% of normal adrenal, and 3.27% of adenoma-associated neovasculature; P.001). Those with more than 25% PSMA-positive vessels were 33 times more likely to be malignant than benign (odds ratio, P.001). Whole-body positron emission tomography-computed tomography imaging showed targeting of anti-PSMA Zr89-J591 to 5/5 of the patient's multiple lung masses with an average measurement of 3.49 ± 1.86 cm and a standardized uptake value of 1.4 ± 0.65 relative to blood pool at 0.8 standardized uptake value.PSMA is significantly overexpressed in ACC neovasculature when compared with normal and benign adrenal tumors. PSMA expression can be used to image ACC metastases in vivo and may be considered as a potential diagnostic and therapeutic target in ACC.

Recent publications have brought attention to the histopathological, immunohistochemical, and molecular aspects of the rare breast tumor resembling the tall cell variant of papillary thyroid carcinoma (BrTC). Nine archived cases of this entity were retrieved, reviewed, and compared with randomly selected tall cell variants of papillary thyroid carcinoma (ThTC). Seven of the BrTC cases as well as 5 cases of solid papillary carcinoma of breast were analyzed by Oncomine next-generation sequencing. BrTC and ThTC were histologically distinguishable by the presence of solid architecture, luminal histiocytes, and reverse polarity in the former, and psammoma bodies, giant cells, and optically clear nuclei in the latter. Sequencing revealed IDH2 R172 single-nucleotide variants in all 7 BrTCs, 6 of which had concurrent PIK3CA mutations. None of the conventional solid papillary carcinomas demonstrated IDH2 mutation. BrTC bears superficial resemblance to other papillary tumors but is unique in terms of histology and molecular profile.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) can be challenging to evaluate histologically. MicroRNAs (miRNAs) are small RNA molecules that often are excellent biomarkers due to their abundance, cell-type and disease stage specificity and stability. To evaluate miRNAs as adjunct tissue markers for classifying and grading well-differentiated GEP-NETs, we generated and compared miRNA expression profiles from four pathological types of GEP-NETs. Using quantitative barcoded small RNA sequencing and state-of-the-art sequence annotation, we generated comprehensive miRNA expression profiles from archived pancreatic, ileal, appendiceal and rectal NETs. Following data preprocessing, we randomly assigned sample profiles to discovery (80%) and validation (20%) sets prior to data mining using machine-learning techniques. High expression analyses indicated that miR-375 was the most abundant individual miRNA and miRNA cistron in all samples. Leveraging prior knowledge that GEP-NET behavior is influenced by embryonic derivation, we developed a dual-layer hierarchical classifier for differentiating GEP-NET types. In the first layer, our classifier discriminated midgut (ileum, appendix) from non-midgut (rectum, pancreas) NETs based on miR-615 and -92b expression. In the second layer, our classifier discriminated ileal from appendiceal NETs based on miR-125b, -192 and -149 expression, and rectal from pancreatic NETs based on miR-429 and -487b expression. Our classifier achieved overall accuracies of 98.5% and 94.4% in discovery and validation sets, respectively. We also found provisional evidence that low- and intermediate-grade pancreatic NETs can be discriminated based on miR-328 expression. GEP-NETs can be reliably classified and potentially graded using a limited panel of miRNA markers, complementing morphological and immunohistochemistry-based approaches to histologic evaluation.

BACKGROUND AND PURPOSE: Skull base chordomas often demonstrate variable MR imaging characteristics, and there has been limited prior research investigating the potential clinical relevance of this variability. The purpose of this retrospective study was to assess the prognostic implications of signal intensity on standard imaging techniques for the biologic behavior of skull base chordomas. MATERIALS AND METHODS: Medical records were retrospectively reviewed for 22 patients with pathologically confirmed skull base chordomas. Clinical data were recorded, including the degree of surgical resection, the presence or absence of radiation therapy, and time to progression/recurrence of the tumor or time without progression/recurrence of the tumor following initial treatment. Pretreatment imaging was reviewed for the presence or absence of enhancement and the T2 signal characteristics. Tumor-to-brain stem signal intensity ratios on T2, precontrast T1, and postcontrast T1 spin-echo sequences were also calculated. Statistical analysis was then performed to assess correlations between imaging characteristics and tumor progression/recurrence. RESULTS: Progression/recurrence of skull base chordomas was seen following surgical resection in 11 of 14 (78.6%) patients with enhancing tumors and in zero of 8 patients with nonenhancing tumors. There was a statistically significant correlation between skull base chordoma enhancement and subsequent tumor progression/recurrence (P < .001), which remained significant after controlling for differences in treatment strategy (P < .001). There was also a correlation between postcontrast T1 signal intensity (as measured by postcontrast T1 tumor-to-brain stem signal intensity ratios) and recurrence/progression (P = .02). While T2 signal intensity was higher in patients without tumor progression (median tumor-to-brain stem signal intensity ratios on T2 = 2.27) than in those with progression (median tumor-to-brain stem signal intensity ratios on T2 = 1.78), this association was not significant (P = .12). CONCLUSIONS: Enhancement of skull base chordomas is a risk factor for tumor progression/recurrence following surgical resection.

Fine-needle aspiration (FNA) is widely accepted in the preoperative management of salivary gland lesions. The proposed Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) aims to standardize reporting terminology. Studies regarding the risk of malignancy (ROM) for the proposed categories continue to evolve. The current retrospective study applied the MSRSGC to assess ROM for salivary gland lesions and focused on the "indeterminate" categories.A total of 627 salivary gland FNA specimens obtained from 2011 through 2016 were retrieved, with follow-up available for 373 cases. The original diagnoses were recategorized using MSRSGC by 2 independent cytopathologists as: 1) non-diagnostic; 2) non-neoplastic; 3) atypia of undetermined significance (AUS); 4a) benign neoplasms; 4b) salivary gland neoplasm of uncertain malignant potential (SUMP); 5) suspicious for malignancy (SFM); and 6) malignant. The ROM and overall ROM for each diagnostic category were determined, with characterization of "indeterminate" (AUS, SUMP, and SFM) lesions.There was near-perfect agreement regarding categorization (626 of 627 cases; 99.8%) between the 2 cytopathologists, with discordance observed for 1 case. The sensitivity, specificity, negative predictive value, and positive predictive value of salivary gland FNA specimens at the study institution were 79%, 98%, 94%, and 92%, respectively. The ROM for non-diagnostic, non-neoplastic, benign neoplasms, AUS, SUMP, SFM, and malignant were 6.7%, 7.1%, 38.9%, 5.0%, 34.2%, 92.9%, and 92.3%, respectively. The indeterminate category had an overall ROM of 47.1%.The MSRSGC is a valuable tool that can help to standardize reporting and stratify cases preoperatively. Having a better understanding of the indeterminate diagnoses will help further refine risk classification criteria. Cancer Cytopathol 2018. © 2018 American Cancer Society.

Background Inhibition of the interaction of programmed death 1 with programmed death ligand 1 and 2 has been used successfully for treatment of multiple advanced cancers, but expression has not been studied in adrenocortical carcinoma. In this study, we investigated programmed death ligand 1 and 2 expression in adrenocortical carcinoma to determine the potential usefulness of checkpoint inhibitors in these malignant neoplasms. Methods A total of 56 tissue samples from patients with adrenocortical carcinoma (34) and benign adrenal tissues (22) were identified. Immunohistochemistry was performed for programmed death ligand 1, programmed death ligand 2, and CD8 and scored for membranous staining on adrenal and stromal tissue according to the immunoreactive score and absolute percentage, respectively. Descriptive statistics, a Mann-Whitney U test, and Fisher exact tests were calculated. Results In total, 15 adrenocortical carcinoma (44%) stained positive for programmed death ligand 2 and 1 adrenocortical carcinoma for programmed death ligand 1 (P = .03). Adrenocortical carcinoma samples were more likely to express programmed death ligand 2 on tumor cells or in stromal tissues than benign samples (OR = 2.3, P = .03). There was no relationship between programmed death ligand 2 and CD8 expression (P = .08). There were also no relationships between programmed death ligand 2 or CD8 expression and tumor characteristics. Conclusion Programmed death ligand 2, but not programmed death ligand 1, is expressed commonly in adrenocortical carcinoma samples. The utility of certain checkpoint inhibitors should, therefore, be evaluated in further studies.

Background Head and neck squamous cell carcinoma (HNSCC) is a cancer that is characterized by its high morbidity and mortality rates. While tobacco use and alcohol consumption are 2 major contributing factors for HNSCC carcinogenesis, how the combination of tobacco and alcohol increases HNSCC risk is not understood. Methods We combined the 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis and Meadows–Cook alcohol mouse models to elucidate the molecular events and to identify the novel biomarkers associated with oral cancer development. Results By genome-wide RNA-seq of tongue samples (3 mice per group), we identified changes in transcripts that mediate alcohol metabolism and oxidative stress (Aldh2, Aldh1a3, Adh1, Adh7, and Cyp2a5) in mice treated with 4-NQO followed by ethanol (4-NQO/EtOH) as compared to the vehicle control/untreated (V.C./Untr.) samples. We measured major, global increases in specific histone acetylation and methylation epigenetic marks (H3K27ac, H3K9/14ac, H3K27me3, and H3K9me3) in the oral cavities of V.C./EtOH, 4-NQO/Untr., and 4-NQO/EtOH treatment groups compared to the V.C./Untr. group. We detected changes in histone epigenetic marks near regulatory regions of genes involved in ethanol metabolism by chromatin immunoprecipitation. For instance, the Aldh2 promoter showed increased H3K27me3 marks, and Aldh2 mRNA levels were reduced by 10-fold in 4NQO/EtOH versus V.C./Untr. tongue samples. 4-NQO/EtOH treatment also caused increases in markers of oxidative stress, including 4-HNE, MCT4/SLC16a3, and TOM20, as measured by immunohistochemistry. Conclusions We delineate a mechanism by which 4-NQO and ethanol can regulate gene expression during the development of HNSCC and suggest that histone epigenetic marks and oxidative stress markers could be the novel biomarkers and targets for the prevention of HNSCC.

// Xiao-Han Tang 1 , Alison M. Urvalek 1,* , Kwame Osei-Sarfo 1,* , Tuo Zhang 2 , Theresa Scognamiglio 3 and Lorraine J. Gudas 1 1 Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA 2 Genomics Resources Core Facility, Weill Cornell Medical College, New York, NY, USA 3 Department of Pathology, Weill Cornell Medical College, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Lorraine J. Gudas, email: // Keywords : cancer diagnosis, squamous cell carcinoma, tongue lesions, RNA-seq Received : February 10, 2015 Accepted : May 31, 2015 Published : June 10, 2015 Abstract We compared the changes in global gene expression between an early stage (the termination of the carcinogen treatment and prior to the appearance of frank tumors) and a late stage (frank squamous cell carcinoma (SCC)) of tongue carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral cavity and esophageal squamous cell carcinoma. Gene ontology and pathway analyses show that increases in “cell cycle progression” and “degradation of basement membrane and ECM pathways” are early events during SCC carcinogenesis and that changes in these pathways are even greater in the actual tumors. Myc, NFκB complex (NFKB1/RELA), and FOS transcription networks are the major transcriptional networks induced in early stage tongue carcinogenesis. Decreases in metabolism pathways, such as in “tricarboxylic acid cycle” and “oxidative phosphorylation”, occurred only in the squamous cell carcinomas and not in the early stages of carcinogenesis. We detected increases in ALDH1A3, PTGS2, and KRT1 transcripts in both the early and late stages of carcinogenesis. The identification of the transcripts and pathways that change at an early stage of carcinogenesis provides potentially useful information for early diagnosis and for prevention strategies for human tongue squamous cell carcinomas.

Background/Aims: Preoperative identification of malignant parotid lesions remains challenging, and thus, some surgeons use frozen section (FS) to assist them in their decision making. We evaluated the pathologic and cost benefit of FS after fine-needle aspiration (FNA) at our institution. Methods: We assessed medical data for 260 patients undergoing parotidectomy with FS. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated for radiology, FNA, and FS. Results: The sensitivities, specificities, and accuracies of FNA and FS were 75.0, 96.4, and 93.2%, and 75.0, 100, and 96.8%, respectively. FS detected 0% of FNA false negatives and 80% of false positives. The additional pathology charge for FS alone per correctly identified benign lesion after a positive FNA was USD 1,443. Conclusion: FNA and FS are more reliable in the prognostication of the final pathology than radiology. At our center, FS appears to be of limited clinical use after benign FNAs, but may be more useful after positive, indeterminate, and nondiagnostic FNAs.

Background. Altered miRNA expression and down-regulation of Dicer has been shown in various cancers. We investigated Dicer expression and global miRNA environment in correlation with malignant features of thyroid tumors. Methods. Dicer gene expression was assessed for 22 normal thyroids, 16 follicular adenomas, 28 papillary thyroid cancers (PTCs), 10 tall-cell variants of PTC, 11 follicular variants of PTC, as well as the four thyroid cell lines BCPAP, TPC1, KTC1, and TAD2 via quantitative polymerase chain reaction. BRAF (V600E) mutation screening was completed for 31 neoplasms. Next-generation sequencing was performed on a subset of PTC and normal thyroid. Protein levels were assessed via Western blotting and immunohistochemistry. Results. Dicer mRNA was down-regulated in malignant thyroid samples and cell lines compared with normal tissues, benign neoplasms, and the fetal cell line TAD2. Decreased Dicer gene expression in malignant tissues was correlated greatly with aggressive features: extrathyroidal extension, angiolymphatic invasion, multifocality, lymph node and distant metastasis, recurrence, and BRAF (V600E) mutation. Conversely, increased levels of Dicer protein were observed in malignant tissues and cell lines. Sequencing yielded 19 differentially expressed miRNAs. Eight samples had a nonsignificant a global down-regulation in malignant tissues. Conclusion. Dysregulation of Dicer and possibly altered expression of miRNAs are associated with aggressive features in thyroid cancers. These findings suggest that disruption in normal miRNA processing involving Dicer may play a role in thyroid cancer progression. (Surgery 2014;156:1342-50.)

In 2006, a multidisciplinary thyroid conference (MDTC) was implemented to better plan management of thyroid cancer patients at our institution. This study assessed the clinical impact of a MDTC on radioactive iodine (RAI) treatment patterns. A prospective database (2003–2014) collected patient and tumor characteristics, RAI doses, and tumor recurrences. Patients treated with total thyroidectomy for differentiated thyroid carcinoma ≥1 cm were stratified based on American Thyroid Association (ATA) risk classification. RAI regimens were compared before initiation of MDTC (2003–2005, n = 88), after establishment of MDTC (2007–2009, n = 95), and after the release of 2009 ATA guidelines (2011–2014, n = 181). RAI doses were defined as low (≤75 mCi), intermediate (76–150 mCi), and high (>150 mCi). There was a significant decrease in the number of patients who received high-dose RAI after implementation of MDTC compared to before initiation of MDTC in the intermediate and high-risk patient groups (p = 0.04 and p

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein receptor, is highly expressed in prostate cancer and in the tumor neovasculature of colon, breast, and adrenocortical tumors. Here, we analyzed PSMA expression in the neovasculature of various thyroid cancer subtypes and assessed whether PSMA expression is correlated with aggressive behavior. From a prospectively maintained database, we evaluated 91 samples from 68 patients, including 37 primary differentiated thyroid cancers (DTCs) [11 classic papillary (cPTC), 9 follicular-variant (FvPTC), 11 follicular (FTC), 6 radioactive iodine-refractory (RAIR)], 5 anaplastic (ATC) carcinomas, 9 distant and 12 lymph node metastases, 21 benign thyroid nodules, and 7 normal thyroid specimens. Formalin-fixed paraffin-embedded tissue blocks were immunostained for vascular endothelial marker CD31 and PSMA with proper controls. PSMA expression was not detected in normal thyroid tissue. DTC tumors demonstrated a significantly higher PSMA expression, in regard to both intensity and percentage of vessels stained, than benign tumors (p

Background In 2016, encapsulated follicular variant of papillary thyroid carcinoma without invasion was renamed "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) in order to reduce overtreatment of this indolent tumor. However, many endocrinologists remain uneasy about managing large (≥4 cm) NIFTP conservatively without radioactive iodine (RAI) therapy. The objectives of this study are to characterize the clinicopathologic characteristics and outcome of large NIFTP in order to assist therapeutic decision making. Methods The pathology databases of four tertiary hospitals were searched for large (≥4 cm) NIFTP. Cases with separate foci of carcinoma were excluded. Seventy-nine cases fulfilled the inclusion criteria. Among them, 56 (71%) had at least two years of clinical follow-up (FU), and 49 (62%) had four or more years of FU. The clinicopathologic characteristics were reviewed and documented by four endocrine pathologists. Results The median size of the NIFTP was 4.5 cm (range 4.0-8.0 cm). The entire capsule was sampled in 50 (63%) tumors, while in the remaining 29 (37%) cases, it was submitted representatively, with a median of 2.1 blocks per centimeter of tumor examined. Large NIFTP had a female preponderance with a male:female ratio of 1:1.8, and presented at a median age of 49 years. There were no lymph node metastases at diagnosis in any of the patients, and none of the patients (n = 25) in whom nodal tissue was available for microscopic examination had positive findings. Twenty-six (33%) underwent thyroid lobectomy alone, and 37 (47%) did not receive RAI ablation. No recurrence was observed in the entire cohort, including all 32 patients with two or more years of FU who did not receive RAI therapy (median FU: 6.7 years). Among patients with four or more years of FU, all 25 individuals without RAI therapy did not recur, with a median FU of 11.2 years. Patients with a larger tumor size tended to receive postoperative RAI ablation (p = 0.001). Conclusions Similar to their small counterparts, large NIFTP appear to have an extremely low risk of recurrence (zero in this cohort), even when treated conservatively without RAI therapy. Surgical treatment alone, including lobectomy, appears to be adequate for large NIFTP.

We reclassified 179 cases of the follicular variant (FV) of papillary thyroid carcinoma (PTC) into 72 (40.2%) noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 37 (20.7%) encapsulated FVPTC with invasion (EFVPTC), and 70 (39.1%) infiltrative FVPTC (IFVPTC) without a capsule. In the NIFTP group, 5.6% cytology were hypercellular and the remainder low to moderate cellularity. PTC nuclei were absent in 18%, focally present in 37.5%, and diffusely present in 44.4%. In the EFVPTC group, 8.1% cytology were hypercellular and the reminder low to moderate cellularity. PTC nuclei were absent in 24.3%, focally present in 29.7% and diffusely present in 45.9%. In IFVPTC group, 24.3% cytology were hypercellular and the reminder low to moderate cellularity. PTC nuclei were diffusely present in 88.6% and focally present in 11.4%. The ultrasound findings for NIFTP and minimally invasive EFVPTC typically demonstrated a circumscribed oval/round nodule with a hypoechoic rim, and the Doppler was mostly hypervascular. The ultrasound findings for overtly invasive EFVPTC typically showed a round/oval nodule with irregular margins and the Doppler was mostly hypervascular. The ultrasound findings for IFVPTC group showed at least one of the malignant gray-scale features: markedly hypoechoic, taller-than-wide, microcalcifications or blurred margins. The Doppler in this group was mostly avascular. An algorithm is proposed to triage thyroid FNA based on different scenarios of the sampled cells, interpreted in the context of ultrasound features with histology outcomes. Five composite ultrasound-cytology-histology figures illustrate NIFTP, IFVPTC, and IFVPTC with intratumoral fibrosis, microfollicular EFVPTC and normofollicular EFVPTC. Diagn. Cytopathol. 2017;45:533-541. © 2017 Wiley Periodicals, Inc.

Cancer/testis (CT) antigens, normally only expressed in germ cells of adult testis, can be activated in malignancy as tumor-specific antigens. The potential value of CT antigens as biomarkers in the evaluation of mucosal squamous precursor lesions of the head and neck has not been investigated. The expression of 8 CT antigens (MAGE-A, GAGE, NY-ESO-1, CT7, CT10, SAGE1, CT45 and NXF2) in 76 cases of invasive head and neck squamous cell carcinoma (SCC) was evaluated immunohistochemically. 65 mucosal biopsies of squamous dysplasia and 55 squamous papillomas with dysplasia were analyzed for 6 CT antigens, using an antibody cocktail. Of invasive SCC, 66% (50/76) expressed at least one CT antigen, most commonly MAGE-A (47%). Among the biopsies, only 1 of 55 squamous papillomas was CT-positive, whereas 8 of 65 (12%) squamous dysplasia lesions were CT-positive. These 8 CT-positive biopsies were from 6 patients, 3 of which had concurrent or subsequent SCC. CT antigens are frequently expressed in head and neck SCC; however, there was no difference in the clinicopathological characteristics or behavior of CT-positive tumors compared to CT-negative tumors. The usefulness of CT antigens as positive predictors for SCC in squamous dysplasia biopsies remains to be determined by long-term follow-up in larger cohorts.

Nasal pyogenic granuloma (lobular capillary hemangioma) is a rare benign tumor of unclear etiology. This study describes the clinical characteristics and possible etiologic factors in a series of patients with pyogenic granuloma.All adult patients with a histologic diagnosis of pyogenic granuloma of the nasal cavity were identified between January 2005 and May 2015. A total of 128 charts were reviewed retrospectively for age, gender, clinical presentation, location of lesion, and medical history.Thirty-eight patients were identified including 13 (34%) men and 25 (66%) women with a mean age of 45 years (range, 18-80 years). The majority (92%) presented with epistaxis and 76% had the lesion located on the septum. All patients had the lesion surgically excised with final pathology confirming pyogenic granuloma. Out of 38 patients, 15 (40%) had a concurrent history of altered hormonal activity (due to hormone therapy, pregnancy, or tumor), and 7 (18%) had prior injury to the nasal cavity.This study is 1 of the largest case series of nasal pyogenic granuloma to be reported. In this series, this disease process was seen in approximately twice as many women as compared to men. Our data further supports prior studies, suggesting that altered hormonal activity and nasal injury may increase the risk of developing nasal pyogenic granuloma.

Sulcus vocalis is an epithelial invagination adherent to deep tissues of the vocal fold. Traditionally, dysphonia is believed to result from attenuation or absence of lamina propria and consequent alteration of mucosal dynamics. This conception does not account for several clinical features of the lesion, most notably inflammation. The goal of this study is to elucidate the clinical nature of sulcus by re-examination of its histopathology.Retrospective review.Clinical features, including stroboscopic examination, and hematoxylin and eosin sections of 19 lesions in 15 patients who underwent surgery were reviewed.Epithelial change was found uniformly in all specimens, consisting of parakeratosis (78.9%), epithelial thickening (77.8%), dyskeratosis (63.2%), basement membrane thickening (47%), epithelial inflammation (52.6%), and retention of keratin debris (36.8%). In contrast, submucosal findings were less prevalent. Seven patients (36.8%) had essentially normal subepithelial tissues. Submucosal inflammation was present in seven (36.8%), and other submucosal changes in four of 17 (23.5%). Clinical characteristics demonstrated general improvement after surgical intervention in most cases.Sulcus vocalis appears to have an important component of epithelial pathology, with especially high prevalence of parakeratosis, dyskeratosis, and epithelial thickening. Clinical changes may result from prominent perilesional inflammation related to epithelial changes instead of or in addition to any alteration of the lamina propria. Surgical treatment, when necessary, should refocus on removal of pathologic epithelium as a source of inflammation independent of reconstructive considerations.4 Laryngoscope, 126:1397-1403, 2016.

Papillary thyroid carcinoma (PTC) is a common malignancy with multiple variants, some of which are rarely encountered in routine surgical pathology practice. PTC with exuberant nodular fasciitis–like stroma (PTC-NFS) or PTC with fibromatosis-like stroma (PTC-FMS) is one such variant. We report a case of PTC-NFS, a tumor characterized by an abundant stromal component with an intervening epithelial component with the typical morphologic features of conventional PTC. We review the literature on this rare variant, discuss the importance of a thorough search for epithelial components within any fibroproliferative lesion of the thyroid, and address the diagnostic difficulties created by the tumor’s extensive stromal component.

We evaluated HBME1, galectin-3 (GAL3), cytokeratin (CK)19, and a new anti-CITED1 antibody in 127 follicular adenoma (FA) and papillary thyroid carcinoma (PTC) cases. The findings were used to evaluate 11 diagnostically challenging encapsulated follicular lesions with questionable features of PTC (FL/QPTC). All 4 markers showed higher expression in PTC than FA. HBME1 was the most specific (96%), whereas CK19 was the most sensitive (96%). In addition, 100% specificity was seen with coexpression of HBME1/CK19. Negative expression of all 4 markers was 97% specific for FA. GAL3 and CITED1, less useful individually, could help in selective cases. FL/QPTC showed heterogeneous, often intermediate, staining patterns, implying that some FL/QPTCs may be biologically borderline lesions or represent a biologic spectrum of PTC. These antibodies can have a confirmatory role in distinguishing the follicular variant of PTC and FA. For FL/QPTC, these antibodies are helpful in some cases, their limitation perhaps suggesting the biologic ambiguity of these lesions.

Esophageal squamous cell carcinomas (ESCCs) are very common, aggressive tumors, and are often associated with alcohol and tobacco abuse. Because ESCCs exhibit high recurrence rates and are diagnosed at late stages, identification of prognostic and drug targets for prevention and treatment is critical. We used the 4-nitroquinoline-1-oxide (4-NQO) murine model of oral carcinogenesis and the Meadows-Cook model of alcohol abuse to assess changes in the expression of molecular markers during the initial stages of ESCC. Combining these two models, which mimic chronic alcohol and tobacco abuse in humans, we detected increased cellular proliferation (EGFR and Ki67 expression), increased canonical Wnt signaling and downstream elements (β-catenin, FoxM1, and S100a4 protein levels), changes in cellular adhesive properties (reduced E-cadherin in the basal layer of the esophageal epithelium), and increased levels of phosphorylated ERK1/2 and p38. Additionally, we found that treatment with ethanol alone increased the numbers of epithelial cells expressing solute carrier family 2 (facilitated glucose transporter, member 1) (SLC2A1) and carbonic anhydrase IX (CAIX), and increased the phosphorylation of p38. Thus, we identified both 4-NQO- and ethanol-specific targets in the initial stages of esophageal carcinogenesis, which should lead to the development of potential markers and therapeutic targets for human ESCC.

The solid variant of papillary thyroid carcinoma (SVPTC) comprises approximately 3% of thyroid cancers, and there are conflicting reports about its behavior in the literature. The cytology of SVPTC is limited to 3 single case reports, a review article, and a monograph. We present the first cytologic study of SVPTC.Fine-needle aspiration smears obtained with ultrasound guidance from 13 patients with histologically pure SVPTC were reviewed, and the cytologic features recorded. Ultrasound images were retrieved from radiology and were correlated with low-power histology images. Intratumor vascularity on Doppler imaging was correlated with cellularity in cytology samples.Three cytomorphologic patterns of SVPTC were identified: cohesive, syncytial-type tissue fragments; microfollicles/trabeculae; and dyshesive single cells. All 3 SVPTCs in the first group were encapsulated without invasion. Two of 6 SVPTCs in the second group had a single lymph node metastasis; 4 were encapsulated, and 2 had pushing borders. Ultrasound images in the first and second SVPTC groups were similar, with the majority revealing a well defined, solid nodule with minimal intranodular vascularity. All 4 SVPTCs in the third group had infiltrative borders; and, with the exception of one 0.8-cm tumor, all had multiple lymph node metastases. Ultrasound in the third group revealed irregular borders. RET/PTC1 and RET/PTC3 mutations were found in 2 cases of the third group.SVPTCs are heterogeneous tumors. The cohesive, syncytial tissue-fragment pattern can be recognized as SVPTC in smears and is associated with encapsulation and indolent behavior. The microfollicular/trabecular pattern is indistinguishable from that of the follicular variant of papillary thyroid carcinoma and has intermediate behavior. The dyshesive single-cell pattern correlates with infiltrative tumor growth and may not be unique to SVPTC.

There are three subtypes of follicular variant papillary thyroid carcinoma (fvPTC): completely encapsulated, well circumscribed, and infiltrative. While infiltrative tumors are more aggressive than completely encapsulated, controversy exists regarding management of fvPTC subtypes. We compared the clinicopathologic features of fvPTC subtypes to those of classic PTC (cPTC) to help guide fvPTC management, using cPTC as a reference. A retrospective review was performed on 316 patients with PTC treated at a single institution from 2004 to 2011. There were 197 cPTC and 119 fvPTC tumors, including completely encapsulated (n = 46), well circumscribed (n = 46), and infiltrative (n = 27). Clinicopathologic data were compared between groups. fvPTC patients had larger tumors than cPTC patients (1.6 cm vs. 1.2 cm, p = 0.001), but age, sex, and family history did not differ. Thirty-one percent of cPTC tumors had extrathyroidal extension compared to 0 % of completely encapsulated, 0 % of well-circumscribed, and 52 % of infiltrative fvPTC tumors (p

We investigated the effects of bexarotene (a retinoid X receptor agonist), CD1530 (a retinoic acid receptor γ selective agonist), and the combination of these two drugs for the prevention of oral carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral-cavity and esophageal squamous-cell carcinoma previously generated in our laboratory. We observed decreased numbers of neoplastic tongue lesions and reduced lesion severity in the 4-NQO plus CD1530 (4N+C) and 4-NQO plus bexarotene plus CD1530 (4N+B+C) groups compared with the 4-NQO group. RNA-Seq analyses showed increases in transcripts in cell proliferation/cell cycle progression pathways in the 4-NQO vs. the untreated group. In addition, β-catenin and matrix metallopeptidase 9 (MMP9) protein levels and reactive oxygen species (ROS), as assessed by 4-hydroxynonenal (4-HNE) staining, were elevated in tongue tissues 17 wk after the termination of the 4-NQO treatment. The 4N+B, 4N+C, and 4N+B+C groups showed dramatically lower levels of β-catenin, MMP9, and 4-HNE staining compared with the 4-NQO group. The major reduction in 4-HNE staining in the retinoid treatment groups suggests a novel mechanism of action, reduction of ROS, by which bexarotene and CD1530 inhibit carcinogenesis.

Amyloid deposits are unexpected in salivary gland tumors. A 60-year-old woman presented with a 1.8 cm, slow-growing parotid mass. Both fine-needle aspiration and frozen section were misinterpreted as pleomorphic adenoma. The final pathology was amyloid-rich low grade adenocarcinoma of the parotid gland. The aspirates consisted of three components: mucin, amyloid, and tumor cells. The mucin was unusually thick, stringy, and metachromatic. The amyloid presented as innumerable concretions scattered solitarily or in small jigsaw puzzle-like aggregates, individually wrapped by tumor cells. The tumor cells had bland oval nuclei and scant-to-abundant cytoplasm, arranged in loosely cohesive small sheets. On histology, the tumor cells were arranged in interconnecting monolayered glands of a wide range of size with small patches of cellular regions composed of plump tumor cells. As the luminal mucin and amyloid deposits enlarged, the lining tumor cells became thin and flat. The glandular lumen molded amyloid concretions into different shapes and sizes. Atrophic or pyknotic tumor cells outlined the amyloid concretions with concentric laminations, reminiscent of corpora amylacea. Alcian blue positive luminal mucin, associated with newly formed amyloid, was present in mucinous regions of the tumor. This is the first description of cytologic features of amyloid-rich low grade adenocarcinoma of the parotid and the second case in the pathology literature. The literature of amyloid-rich tumors was reviewed and the implication of the presence of abundant amyloid on the death of tumor cells suggested.

Background The tall cell (TC) variant of papillary thyroid carcinoma (PTC) is more aggressive than classic PTC, but the percentage of TC necessary to diagnose this variant has been debated. We aimed to better determine what percentage of TC correlates with a more aggressive phenotype. Methods Cases from 2005 to 2010 that were the TC variant of PTC or PTC with TC features were identified and compared with classic PTCs. All cases were reviewed to determine what percent, if any, of the tumors consisted of TC. Results One hundred forty-one cases of PTC were reviewed. Eighty-three cases had some TC component, and 58 cases had none. There were no differences in patient demographics. Tumors with ≥10% TC had more extrathyroidal extension, angiolymphatic invasion, positive surgical margin, and lymph node involvement than classic PTC. There were more recurrences in patients with ≥10% TC, but this was not significant. Similar findings were also observed with increasing percentages of TC. Conclusion The aggressive features conferred by the presence of TC in PTCs occur with as little as 10% TC and are maintained with increasing percentages. Therefore, TC in a PTC should be reported if they comprise at least 10% of the tumor.

The advent of sentinel lymph node (SLN) biopsy for breast cancer has had a considerable impact on clinical oncology. The SLN procedure has been widely adopted, but major clinical issues remain unresolved pending the outcome of randomized trials. Pathologic handling of SLNs is less controversial; however, significant pitfalls in their handling have been reported. Herein we report two additional uncommon situations that demonstrate the need for the utmost vigilance in both the macroscopic examination (i.e., trimming of “positive” nodal tissue) and microscopic examination (i.e., presence of nevus cell aggregates mimicking metastatic carcinoma) of SLN specimens.

BACKGROUND: Preoperative B-type Raf kinase Val600Glu mutation, or BRAF(V600E), analysis has been proposed as a tool to guide initial surgery for indeterminate thyroid nodules. This study sought to determine if cytologic markers of malignancy are associated with the BRAF(V600E) mutation and if preoperative BRAF(V600E) testing would alter the initial management of patients with indeterminate nodules. METHODS: Patients who underwent surgery for a thyroid nodule between 2003 and 2012 at a tertiary care center were prospectively enrolled. Stored nodule samples were retrospectively genotyped for the BRAF(V600E) mutation. BRAF(V600E) status, demographics, cytologic and histopathologic findings, and choice of initial surgery were examined. RESULTS: A total of 960 patients were enrolled, of which 310 (32%) had an indeterminate nodule. The BRAF(V600E) mutation was identified in 13 patients (4%), 12 of whom had either cytologic atypia or were Bethesda category V. Three percent of Bethesda category III or IV nodules that were malignant harbored the mutation compared with 42% of Bethesda category V malignancies. Nuclear grooves (P = .030), pseudoinclusions (P < .001), and oval nuclei (P = .022) were all more common among BRAF(V600E) mutants. The sensitivities of using BRAF testing alone, cytologic atypia/Bethesda category V classification, or both, were 15%, 73%, and 76%, respectively. Twelve of the 13 BRAF(V600E) mutants had total thyroidectomies initially due to worrisome cytologic features, and therefore the initial management of only one patient would have been altered if BRAF(V600E) testing had been performed preoperatively. CONCLUSIONS: Preoperative mutation screening for BRAF(V600E) does not meaningfully improve risk stratification and is unlikely to alter the initial management of patients with indeterminate nodules. Cancer 2013. © 2012 American Cancer Society.

Primary thyroid tumors with extensive clear cell changes are extremely rare. More than 10,000 ultrasound-guided thyroid fine needle aspirations examined over a period of 17 years by the first author, only one of the 530 (0.2%) papillary thyroid carcinomas (PTCs) and three of the 42 (7.1%) follicular thyroid carcinomas (FTCs) had extensive cytoplasmic clearing. Thyroidectomies were performed for these cases at four different hospitals in New York City. Final pathology was available for review in each of the four cases. Histology showed a 1.5-cm PTC with prominent hobnail features and clear cell change in a 31-year-old woman, a 4.3-cm FTC without angioinvasion in a 31-year-old woman, a 4.5-cm angioinvasive FTC in a 45-year-old man, and a 2-cm FTC with extensive angioinvasion in a 41-year-old woman with McCune-Albright syndrome (previously published). On ultrasound, the FTCs were solid circumscribed nodules and the PTC had an irregular margin. In these cases, the etiology for the cytoplasmic clearing included accumulation of glycogen in the PTC, accumulation of vesicles in two of the FTCs, and accumulation of lipid droplets in a FTC with extensive angioinvasion. Review of the cytologic literature showed 17 cases of follicular-derived thyroid tumors with extensive clear cell change. To the best of our knowledge, this is the first cytologic report of PTC with hobnail features and extensive clear cell change.

Purpose: Indeterminate thyroid lesions on fine needle aspiration (FNA) harbor malignancy in about 25% of cases. Hemi- or total thyroidectomy has, therefore, been routinely advocated for definitive diagnosis. In this study, we analyzed miRNA expression in indeterminate FNA samples and determined its prognostic effects on final pathologic diagnosis. Experimental Design: A predictive model was derived using 29 ex vivo indeterminate thyroid lesions on FNA to differentiate malignant from benign tumors at a tertiary referral center and validated on an independent set of 72 prospectively collected in vivo FNA samples. Expression levels of miR-222, miR-328, miR-197, miR-21, miR-181a, and miR-146b were determined using reverse transcriptase PCR. A statistical model was developed using the support vector machine (SVM) approach. Results: A SVM model with four miRNAs (miR-222, miR-328, miR-197, and miR-21) was initially estimated to have 86% predictive accuracy using cross-validation. When applied to the 72 independent in vivo validation samples, performance was actually better than predicted with a sensitivity of 100% and specificity of 86%, for a predictive accuracy of 90% in differentiating malignant from benign indeterminate lesions. When Hurthle cell lesions were excluded, overall accuracy improved to 97% with 100% sensitivity and 95% specificity. Conclusions: This study shows that that the expression of miR-222, miR-328, miR-197, and miR-21 combined in a predictive model is accurate at differentiating malignant from benign indeterminate thyroid lesions on FNA. These findings suggest that FNA miRNA analysis could be a useful adjunct in the management algorithm of patients with thyroid nodules. Clin Cancer Res; 18(7); 2032–8. ©2012 AACR.

Endoscopic thyroidectomy is gaining popularity, particularly in Asian countries, as an alternative to conventional cervical thyroidectomy. Multiple large case series have been published that confirm the feasibility and safety of this procedure compared to conventional methods. However, no data are available that demonstrate long-term oncologic outcomes or complications.A patient who underwent transaxillary gas insufflation thyroidectomy for a follicular neoplasm presented to the authors with a mass along the operative tract 31 months after her first surgery. The mass was found to be a recurrence of the thyroid neoplasm.Since this is a relatively new procedure, the potential complications differ from those of conventional cervical thyroidectomy. Further studies are needed to determine size limitations on nodules considered for endoscopic resection.When performing these procedures, care should be taken to ensure that the specimen is removed entirely and in one piece. Long-term outcomes data for endoscopic thyroidectomy are warranted prior to this becoming an acceptable standard of care for thyroid surgery.

Objective: Mucin-producing thyroid tumors are extremely rare. Nonetheless, we have encountered three such cases in fine-needle aspiration. We report the cytologic and histologic findings and review the literature. Study Design: Cytologic features were studied on direct smears using Romanovsky stain to detect background substance and Papanicolaou stain to analyze nuclear and cytoplasmic features. The cytologic features were correlated with histology. Mucin was demonstrated by mucicarmine, Alcian Blue/PAS, and Alcian Blue (pH 2.5). Results: The cytologic features related to mucin include: (1) thick luminal mucin globules and signet ring cells aspirated from an 83-year-old woman with a 3-cm signet ring cell follicular adenoma, (2) abundant fluffy mucin containing signet ring cells in cohesive fragments aspirated from a 75-year-old man with the bilateral signet ring cell follicular variant of papillary carcinoma, which is the first case in the English literature, and (3) abundant thin mucoid mucin aspirated from the lymph node of an 86-year-old woman with a 5-cm mucinous poorly differentiated thyroid carcinoma. Conclusion: The occurrence of mucin in thyroid fine-needle aspiration does not necessarily indicate metastasis, and the presence of mucin in cervical lymph nodes does not exclude the thyroid gland as a possible primary.

Microarray technology provides a new opportunity to improve the diagnostic accuracy of fine needle aspiration (FNA) in evaluating thyroid nodules. Here, we evaluate whether ex vivo FNA and tissue samples can be used interchangeably in microarray and whether the method of acquisition affects the precision of the gene list that is generated. To assess whether FNA samples provide adequate material for reliable gene expression analysis, paired tissue and FNA samples were collected from 13 thyroid nodules; 7 malignant, 6 benign. RNA was extracted from each specimen, converted to complimentary DNA and hybridized to AffymetrixU-133 GeneChips. Cluster analysis was then performed using 61 genes predetermined to differentiate benign from malignant nodules. Clustering patterns were evaluated using 2-group K-means and hierarchical analysis. Twelve concordant pairs were used to generate differentially expressed genes between the sampling methods. Twenty-five of 26 samples clustered concordantly with the pathologic diagnosis. The sensitivity, specificity, and accuracy were 100%, 100%, and 100% for FNA and 85.7%, 100%, and 92.3% for tissue, respectively. Two-group K-means revealed an adjacent grouping for 12 of 13 pairs. Hierarchical analysis clustered 8 of 13 pairs together. Sixty-seven genes were differentially expressed between FNA and the tissue sampling methods. These genes predominantly represented stromal components and were upregulated in the tissue compared with FNA samples. We conclude that FNA is a reliable alternative to tissue samples in predicting malignancy with microarray.

The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and retinoic acid receptor β2, by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza; 0.25 mg/kg body weight), all-trans retinoic acid (RA; given at 100 μg/kg body weight and 1 mg/kg body weight), and the combination of 5-Aza and the low-dose RA on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model. All the drug treatments were done for 15 weeks after a 10-week 4-NQO treatment. Mice in all drug treatment groups showed decreases in the average numbers of neoplastic tongue lesions. The combination of 5-Aza and RA effectively attenuated tongue lesion severity. Although all drug treatments limited the increase in the percentage of proliferating cell nuclear antigen–positive cells and the decrease in the percentage of p16-positive cells caused by the 4-NQO treatment in mouse tongue epithelial regions without visible lesions and in the neoplastic tongue lesions, the combination of 5-Aza and RA was the most effective. Collectively, our results show that the combination of a DNA demethylating drug and RA has potential as a strategy to reduce oral cavity cancer in this 4-NQO model.

Lecithin:retinol acyltransferase (LRAT) is an enzyme that converts retinol (vitamin A) to retinyl esters. Its expression is often reduced in human cancers, including oral cavity cancers. We investigated the effects of ectopic expression of human lecithin:retinol acyltransferase (LRAT) on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). We targeted human LRAT expression specifically to the basal layer of mouse skin and oral cavity epithelia by using a portion of the human cytokeratin 14 (K14) promoter. High levels of human LRAT transgene transcripts were detected in the tongues and skin of adult transgenic positive (TG+) mice, but not in transgenic negative (TG-) mice. The retinyl ester levels in skin of LRAT TG+ mice were 32% +/- 5.4% greater than those in TG- mice, and topical treatment of the back skin with retinol resulted in greater increases in retinyl esters (from 6.9- to 14.3-fold in different TG+ mice) in TG+ mouse skin than in TG- mouse skin (1.3 fold). While carcinogen (4-NQO) treatment induced multifocal precancerous and cancer lesions in the tongues of both TG positive (n=16) and negative mice (n=22), higher percentages of transgenic positive mice (62.5%) developed more severe tongue lesions (grades 3 and 4) than transgenic negative mice (24.8%) after 4-NQO treatment (p0.05). Carcinogen treatment also resulted in greater percentages of transgenic positive mouse tongues with hyperplasia (71.4%), dysplasia (85.7%, p0.05), and carcinoma (28.6%) than transgenic negative mouse tongues (53.3%, 46.7%, and 20%, respectively). Moreover, we observed higher cyclooxygenase-2 (Cox-2) and lower RARbeta(2) mRNA levels in TG+ mouse tongues as compared to TG- mouse tongues after 4-NQO treatment (p0.05). Taken together, these data show that overexpression of human LRAT specifically in oral basal epithelial cells makes these cells more sensitive to carcinogen induced tumorigenesis.

Functional chemokine receptors are expressed in many malignant tumors, including papillary thyroid carcinoma (PTC). These receptors promote tumor growth and metastasis in response to endogenous chemokines. The purpose of this study was to examine the expression of two chemokine receptors—CXCR4 and CCR7—in a series of PTCs. We hypothesized that CXCR4 and CCR7 would correlate with indicators of tumor aggressiveness, including tumor size, extrathyroidal extension (ETE), angiolymphatic invasion (ALI), and lymph node metastasis. CXCR4 and CCR7, as well as their specific chemokine ligands (CXCL12 and CCL21, respectively), were assessed in 88 PTCs from 65 patients using a semiquantitative measure of immunohistochemical (IHC) staining intensity for each molecule. Staining intensity was compared with clinicopathologic features including patient age, gender, tumor size, multifocality, ETE, ALI, and lymph node metastasis. Differences in CXCR4 and CCR7 mRNA levels were sought in a subset of tumors using gene microarrays and quantitative RT-PCR. [Statistics: t test, Mann-Whitney U test; P

Extrathyroidal extension (ETE) is a risk factor for recurrence of papillary thyroid carcinoma (PTC). Although initial data supporting this was based on gross ETE noted at surgery, current treatment regimens group patients with microscopic ETE-identified only on histopathology-similarly to those with macroscopic (gross) ETE. This study was designed to assess the influence of microscopic ETE on disease recurrence.Retrospective analysis of 212 patients undergoing thyroidectomy for PTC between 1995 and 2004 with minimum 3-year follow-up was conducted.Of 212 patients, 71 had ETE; 32% were macroscopic and 68% microscopic. Patient demographics, tumor variables, and adjuvant therapy were similar between both ETE groups. Recurrence rates were 52% for macroscopic ETE, 21% for microscopic ETE, and 13% without ETE. On multivariate analysis, patients with macroscopic ETE had a 6.4-fold increased relative risk of recurrence compared with patients with microscopic ETE (P.02; 95% confidence interval, 1.6-25.9) and a significantly decreased disease-free survival (DFS). Furthermore, patients with microscopic ETE had neither a significantly increased risk of recurrence nor different DFS compared with patients without ETE.Macroscopic ETE has a higher incidence of disease recurrence than microscopic ETE, implying they should be considered separately when devising adjuvant treatment regimens. The significance of microscopic ETE is undetermined.