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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.

Oral squamous cell carcinoma (oral SCC) is an aggressive disease and despite intensive treatments, 5-year survival rates for patients have remained low in the last 20 years. Enhancer of zeste homolog 2 (EZH2), part of polycomb repressive complex 2 (PRC2), is highly expressed in human oral SCC samples and cell lines and has been associated with greater epithelia-to-mesenchymal transition (EMT), invasion and metastasis. Here, we developed a tamoxifen-regulated, transgenic mouse line (KcEZH2) in which EZH2 is selectively knocked out (KO) in some tongue epithelial basal stem cells (SCs) in adult mice. EZH2 KO SCs do not show the H3K27me3 mark, as assessed by double-label immunofluorescence. We used this mouse line to assess EZH2 actions during oral tumorigenesis with our immunocompetent 4-nitroquinoline 1-oxide model of oral SCC. We report that higher percentages of mice with invasive SCCs and high-grade neoplastic lesions are observed in mice containing EZH2 KO SCs (KcEZH2-2TΔ and KcEZH2-5TΔ mice). Moreover, EZH2 expression does not correlate with the expression of markers of invasive SCCs. Finally, EZH2 KO cells that are E-cadherin+ are present at invasion fronts infiltrating underlying muscle tissue. Our findings indicate that the knockout of EZH2 in basal SCs of tongue epithelia results in more aggressive carcinomas, and this should be considered when targeting EZH2 as a therapeutic strategy.

Objectives Salivary gland acinic cell carcinoma (AciCC) has recognizable cytomorphologic features that can overlap with benign and malignant entities, creating a diagnostic challenge. AciCC harbors a t(4;9) translocation increasing nuclear receptor subfamily 4 group A member 3 (NR4A3) expression, detectable by immunohistochemistry (IHC) on surgical resection (SR). NR4A3 IHC cytology data are limited. Here, we examine NR4A3 IHC on smears, cell blocks (CBs), and SRs of AciCC and its mimickers. Methods Our cohort comprised AciCC (including high-grade transformation), secretory carcinoma, mucoepidermoid carcinoma (MEC), Warthin tumor, pleomorphic adenoma (PA), cellular PA, carcinoma ex-PA, oncocytic carcinoma, oncocytoma, and nodular oncocytosis. NR4A3 IHC (Santa Cruz Biotechnology and Origene antibodies) was positive if more than 5% tumor cells showed nuclear staining. Results Among CBs, 90% of AciCC cases and none of the mimickers expressed NR4A3. Among SRs, 100% of AciCC cases showed diffuse NR4A3, whereas one high-grade MEC expressed focal NR4A3. Concordance was 95% with two antibody clones. Sensitivity, specificity, positive predictive value, and negative predictive value were 90%, 100%, 100%, and 94.7% for CBs and 100%, 98.8%, 92.3%, and 100% for SRs, respectively. NR4A3 immunostaining was demonstrable on smears from an AciCC case. Conclusions NR4A3 IHC can be a robust diagnostic tool to identify AciCC, especially for cytology specimens.

Purpose: Advanced thyroid cancers, including poorly differentiated and anaplastic thyroid cancer (ATC), are lethal malignancies with limited treatment options. The majority of patients with ATC have responded poorly to programmed death 1 (PD1) blockade in early clinical trials. There is a need to explore new treatment options. Experimental Design: We examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of patients with thyroid cancer. Next, we studied the tumor-targeting efficacy and T-cell dynamics of monotherapy and combination treatments of ICAM1-targeting chimeric antigen receptor (CAR) T cells and anti-PD1 antibody in a xenograft model of ATC. Results: Advanced thyroid cancers were associated with increased expression of both ICAM1 and PD-L1 in tumors, and elevated PD1 expression in CD8+ T cells of circulating blood. The expression of ICAM1 and PD-L1 in ATC lines was regulated by the IFNγ–JAK2 signaling pathway. ICAM1-targeted CAR T cells, produced from either healthy donor or patient T cells, in combination with PD1 blockade demonstrated an improved ability to eradicate ICAM1-expressing target tumor cells compared with CAR T treatment alone. PD1 blockade facilitated clearance of PD-L1 high tumor colonies and curtailed excessive CAR T expansion, resulting in rapid tumor clearance and prolonged survival in a mouse model. Conclusions: Targeting two IFNγ-inducible, tumor-associated antigens—ICAM1 and PD-L1—in a complementary manner might be an effective treatment strategy to control advanced thyroid cancers in vivo.

In 2017, the American College of Radiology (ACR) created the Thyroid Imaging, Reporting, and Data System (TI-RADS) to select thyroid nodules for fine-needle aspiration (FNA). The objective of this study is to find out whether ACR TI-RADS is useful in triaging thyroid follicular cells with papillary-like nuclear features obtained by FNA to determine the extent of surgery.The grayscale ultrasound of 76 noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 41 encapsulated (E), and 79 infiltrative (I) follicular variant (FV) of papillary thyroid carcinoma (PTC) were reviewed and assigned TI-RADS points and then suspicion levels.Of the 39 tumors with high suspicion level, 32 were IFVPTC, and seven were EFVPTC. Of the 137 tumors with moderate suspicion level, 47 were IFVPTC, 34 were EFVPTC, and 56 were NIFTP. Of the 19 tumors with mild suspicion level, all were NIFTP. The only tumor with no suspicion was an NIFTP. IFVPTC had a significantly higher suspicion level than EFVPTC and NIFTP (P0.0001). The difference in suspicion level between EFVPTC and NIFTP is not statistically significant. None of the cases of NIFTP in the study had a high suspicion level.The study demonstrates that cytology interpreted in the context of ACR TI-RADS suspicion levels can separate NIFTP from many IFVPTC and a few EFVPTC with overt invasion. PTC could be diagnosed in cytology if cells with papillary-like nuclear features derived from TR5 nodules. The nodules with TR4 or less that yield similar cells require surgical pathology to diagnose FVPTC with microscopic capsular or vascular invasions.

Context.— An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. Objective.— To describe the validation studies as per New York State–Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. Design.— Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. Results.— High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. Conclusions.— This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

Polymorphous adenocarcinoma (PAC) shows histologic diversity with streaming and targetoid features whereas cribriform adenocarcinoma of salivary gland (CASG) demonstrates predominantly cribriform and solid patterns with glomeruloid structures and optically clear nuclei. Opinions diverge on whether CASG represents a separate entity or a variant of PAC. We aimed to assess the level of agreement among 25 expert Head and Neck pathologists in classifying these tumors. Digital slides of 48 cases were reviewed and classified as: PAC, CASG, tumors with ≥50% of papillary architecture (PAP), and tumors with indeterminate features (IND). The consensus diagnoses were correlated with a previously reported molecular alteration. The consensus diagnoses were PAC in 18/48, CASG in16/48, PAP in 3/48, and IND in 11/48. There was a fair interobserver agreement in classifying the tumors (κ= 0.370). The full consensus was achieved in 3 (6%) cases, all of which were classified as PAC. A moderate agreement was reached for PAC (κ= 0.504) and PAP (κ= 0.561), and a fair agreement was reached for CASG (κ= 0.390). IND had only slight diagnostic concordance (κ= 0.091). PAC predominantly harbored PRKD1 hotspot mutation, whereas CASG was associated with fusion involving PRKD1, PRKD2, or PRKD3. However, such molecular events were not exclusive as 7% of PAC had fusion and 13% of CASG had mutation. In conclusion, a fair to moderate interobserver agreement can be achieved in classifying PAC and CASG. However, a subset (23%) showed indeterminate features and was difficult to place along the morphologic spectrum of PAC/CASG among expert pathologists. This may explain the controversy in classifying these tumors.

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4(+) T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.

We retrospectively investigated the Genomic Prostate Score® assay in clinical practice at an urban tertiary care academic center.We reviewed all Genomic Prostate Score results acquired during a 3-year period. Changes in patient NCCN® (National Comprehensive Cancer Network®) risk group, including very low, low, intermediate or high risk, and ultimate management decisions were recorded.Genomic Prostate Score risk stratification was performed in 134 men. According to the NCCN Guidelines®, 31 of the 134 men (23.1%) were at very low risk, 45 (33.6%) were at low risk and 58 (43.3%) were at intermediate risk. After adding the score the risk group changed in 32 of 134 patients (23.9%). The risk group did not change in the 31 men at very low risk. However, in the low risk group the risk changed in 19 of the 45 men (42.2%), including in 15 to very low and in 4 to intermediate risk. Also, in the intermediate risk group the risk changed in 13 of the 58 men (22.4%), including to low in 12 and to high risk in 1. Nine of the 15 men (60%) in whom risk changed from low to very low elected active surveillance. Nine of the 12 patients (75%) at intermediate risk in whom risk changed to low risk elected active surveillance, 2 (16.7%) elected definitive therapy and in 1 (8.3%) the choice was unknown. Of the 45 men at intermediate risk in whom risk was unchanged 28 (62.2%) elected definitive therapy, 12 (26.0%) elected active surveillance and in 5 (11.1%) the choice was unknown. Of the 4 men upgraded from low to intermediate risk after adding the genomic prostate score 2 elected definitive therapy and 2 chose active surveillance.The Genomic Prostate Score has limited clinical usefulness in patients at very low risk since the NCCN risk group did not change. While it may be more useful for men at low and intermediate risk, for 32 (31%) of whose risk group was reclassified, clinical management decisions did not always appear to reflect these changes.

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.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET–rearranged thyroid cancer.

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.

Purpose: Poorly differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet lethal malignancies with limited treatment options. Many malignant tumors, including papillary thyroid cancer (PTC) and ATC, are associated with increased expression of ICAM-1, providing a rationale for utilizing ICAM-1–targeting agents for the treatment of aggressive cancer. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 to leverage adoptive T-cell therapy as a new treatment modality. Experimental Design: ICAM-1 CAR T cells were applied to multiple malignant and nonmalignant target cells to investigate specific target cell death and “off-tumor” toxicity in vitro. In vivo therapeutic efficacy of ICAM-1 CAR T cells was examined in ATC mouse models established from a cell line and patient-derived tumors that rapidly develop systemic metastases. Results: ICAM-1 CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro. Interestingly, although certain ATC cell lines showed heterogeneous levels of ICAM-1 expression, addition of cytotoxic CAR T cells induced increased ICAM-1 expression such that all cell lines became targetable. In mice with systemic ATC, a single administration of ICAM-1 CAR T cells mediated profound tumor killing that resulted in long-term remission and significantly improved survival. Patient-derived ATC cells overexpressed ICAM-1 and were largely eliminated by autologous ICAM-1 CAR T cells in vitro and in animal models. Conclusions: Our findings are the first demonstration of CAR T therapy against both a metastatic, thyroid cancer cell line and advanced ATC patient-derived tumors that exhibit dramatic therapeutic efficacy and survival benefit in animal studies. Clin Cancer Res; 23(24); 7569–83. ©2017 AACR.

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

Context.—The identification of precursor or dysplastic lesions in the thyroid is difficult. Pathology of the C cell has been extensively studied, and the preneoplastic nature of C-cell hyperplasia in the setting of familial medullary thyroid carcinomas is well established. However, the distinction between neoplastic and physiologic/reactive C-cell hyperplasia remains a challenge. Unlike C cells, the existence of a precursor lesion of follicular cell–derived tumors is less well established, and a dysplastic or preneoplastic follicular lesion has not been well defined.Objective.—To discuss putative precursor lesions in the thyroid arising from C cells and follicular epithelial cells.Data Sources.—Data were obtained from a review of the pertinent peer-reviewed literature.Conclusions.—Although the preneoplastic nature of C-cell hyperplasia in the setting of familial medullary thyroid carcinoma is well recognized, the preneoplastic nature/malignant potential of reactive/physiologic C-cell hyperplasia and its role in the development of sporadic, medullary thyroid carcinoma is still unclear. Current data suggest that benign follicular lesions may have malignant potential, and there may be a multifocal progression from benign to malignant. Atypical follicular lesions in the background of chronic lymphocytic thyroiditis may represent dysplastic or premalignant lesions.

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.

Diffuse embryoma is a rare pattern of nonseminomatous germ cell tumor of the testis originally described in 1983. We report a case with this predominant pattern in an 18-year-old male with a painless palpable testicular mass. Although it is relatively common to see a diffuse embryoma pattern focally in mixed nonseminomatous germ cell tumors of the testis, it is rarely the predominant pattern and can represent a diagnostic pitfall on routine hematoxylin and eosin stain. We emphasize the importance of recognizing the individual components within the diffuse embryoma pattern, review the literature, and briefly discuss the ancillary immunohistochemical stains that may be utilized to help support the diagnosis.

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.

// 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

Anti-PD1 antibody has been approved for metastatic squamous cell carcinoma of the head and neck (SCCHN) and objective response rates of approximately 20% have been reported. Defining PD-L1 expression at ≥ 1% tumor cells as positive, PD-L1-positive tumors showed a higher response rate. However, it is unclear whether 1% is the optimal cutoff, and studies on lung cancer suggested 50% cutoff as a stronger predictive biomarker. 96 primary SCCHN from oropharynx and oral cavity and 34 corresponding metastatic lesions were typed for membranous PD-L1 expression. p16 immunohistochemistry was used as a surrogate marker for HPV status in SCCHN from the oropharynx. Fifty-two of 96 (54%) tumors were PD-L1-positive, 72% if PD-L1 expression in tumor-infiltrating immunocytes was also included as positive. Fifteen of 34 primary-metastasis tumor pairs differed in PD-L1 expression, and p16(+) cases more frequently showed PD-L1 expression in immunocytes than p16(−) cases (82 vs. 45%, 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.

Context.—Napsin A is a useful marker for distinguishing primary from metastatic lung tumors. Mucinous lung tumors may be difficult to distinguish from metastatic mucinous tumors. Objectives.—To evaluate napsin A expression in lung and extrapulmonary mucinous tumors on both histology and cytology specimens and to determine napsin A's utility in differentiating primary from metastatic mucinous tumors. Design.—Napsin A immunohistochemistry was performed using a rabbit polyclonal antibody on formalin-fixed, paraffin-embedded surgical and fine-needle aspiration biopsy–derived, paraffin-embedded cell block specimens. Positive expression was defined as coarse, granular, cytoplasmic staining in 10% or more of tumor cells. Results.—Sixteen of 32 mucinous lung tumors (50%) and 16 of 33 extrapulmonary mucinous tumors (48%), including 15 of 18 of gastrointestinal origin (83%), expressed napsin A. Positivity was concordant between surgical and cell block specimens in 5 of 9 cases (56%). In 3 of 4 discordant cases, napsin A expression was detected on the surgical specimen but not the cell block. The cell block material in these cases was paucicellular. Conclusions.—Napsin A shows decreased sensitivity and specificity for mucinous lung tumors and is unlikely to be reliable as a sole immunohistochemical marker of lung origin for such tumors (52% specificity in this study). The high frequency of napsin A expression in gastrointestinal mucinous tumors makes it particularly unreliable in distinguishing metastatic gastrointestinal from primary lung mucinous tumors. However, napsin A expression analysis may facilitate distinguishing mucinous tumors of pulmonary from those of nongastrointestinal origin. Interpretation of napsin A staining may be problematic in mucinous tumor specimens of low cellularity such as cell blocks.

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.

MicroRNA (miRNA) microarray analysis has consistently found altered expression of miRNAs in thyroid tumors, suggesting their roles in thyroid carcinogenesis. To explore whether this differential expression can be used as a diagnostic tool in surgical pathology and fine-needle aspirate (FNA) specimens, the expression of selected miRNA was evaluated by quantitative RT-PCR, using total RNA from 84 formalin-fixed paraffin-embedded tissues and 40 ex vivo aspirate specimens. miRNA from all paraffin-embedded tissues and all but one FNA sample were found to be analyzable, with paraffin sections yielding better miRNA quality. Preliminary analysis of 6 miRNAs in 10 papillary thyroid carcinoma and 10 follicular adenoma identified significant overexpression of miR-146b, -221, and -222 in papillary thyroid carcinoma (P0.02), but not miR-146a, -155, or -187 (P0.08). The expression of these first three miRNAs was examined in a series of 5 normal thyroid, 11 hyperplastic nodules, 24 follicular adenoma, 27 classical papillary thyroid carcinoma, 5 follicular variant papillary thyroid carcinoma, 2 follicular carcinoma, and 10 encapsulated follicular lesions with partial nuclear features of papillary carcinoma. Results showed miR-146b to be most consistently overexpressed in both classical papillary carcinoma and follicular variants, whereas all other groups showed lower expression at a similar level (P0.001 for pair-wise comparisons between papillary carcinoma and all other groups). Follicular lesions with partial features of papillary carcinoma all showed low miR-146b levels similar to other non-papillary carcinoma groups, suggesting that they are biologically distinctive from papillary carcinoma. miR-221 and miR-222 also showed higher expression in papillary carcinoma, but with substantial overlaps with the other groups. When applied to 40 FNA samples of various lesions, only miR-146b and miR-222 persisted as distinguishing markers for papillary carcinoma. We concluded that miRNAs, particularly miR-146b, might potentially be adjunct markers for diagnosing papillary thyroid carcinoma in both FNA and surgical pathology specimens.

Objective: Pulmonary hamartomas have a characteristic heterogeneous radiological appearance. However, when composed predominantly of undifferentiated mesenchymal fibromyxoid component, their homogeneous appearance on computed tomography is indeterminate for malignancy. Rendering an accurate preoperative diagnosis in these cases can alter management. The aim of this study was to determine the incidence and accuracy of cytodiagnosis for hamartomas ‘indeterminate’ by imaging. Methods: We retrospectively reviewed records for hamartomas diagnosed by transthoracic fine needle aspiration (FNA) including immediate impressions and final diagnoses. Cytological features evaluated included the presence of fibromyxoid stroma, bronchioloalveolar cell hyperplasia, fibroadipose tissue, cartilage and smooth muscle. Results: Eighteen (1.3%) hamartomas were identified from 1355 transthoracic FNAs. The immediate impression was hamartoma in 13 (72%), carcinoid in one (6%), mucinous bronchioloalveolar carcinoma in two (11%) and non-diagnostic in two (11%). The final diagnosis of hamartoma in cases diagnosed as carcinoid, mucinous bronchioloalaveolar carcinoma and non-diagnostic on immediate impression was rendered following assessment of all cytological material. Conclusion: Overall, FNAs are highly reliable for diagnosing hamartomas even when composed principally of undifferentiated mesenchymal fibromyxoid stroma, especially with the aid of all available preparations including Diff-Quik smears, Papanicolaou smears, ThinPreps and cell block material.

Benign thyroid tumors account for most nodular thyroid disease. Determination of whether a thyroid nodule is benign or malignant is a major clinical dilemma and underlies the decision to proceed to surgery in many patients. Although the accuracy of thyroid nodule fine-needle aspiration (FNA) has reduced the need for surgery over the years, questions regarding how to follow FNA-designated benign nodules remain unresolved. This is true at least in part because of uncertainty over whether some benign nodules harbor malignant potential.An evidence-based review of recent clinical, pathologic, and molecular data is presented. A summary of data and observations from our own experience is also provided.Review of our recent 10-year experience indicates that 2% of thyroid malignancies arise within a preexisting benign thyroid nodule. In addition, both cytologic and molecular tumor markers, including Gal-3, CITED1, HBME-1, Ras, RET/PTC, and PAX8/PPAR gamma, have been identified in some histopathologically classified benign nodules. Gene expression profiling suggests that follicular adenomas and Hürthle cell adenomas have similarities to both benign and malignant tumors, suggesting that some of these tumors are premalignant. In addition, 10% of surgically excised follicular tumors are encapsulated follicular lesions with nuclear atypia, which have been termed "well-differentiated tumors of uncertain malignant potential." The data available suggest that these tumors could be precursors to carcinoma.Some benign thyroid nodules have malignant potential. Further molecular testing of these tumors can shed light on the pathogenesis of early malignant transformation.

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.

Riedel thyroiditis is a rare fibrosing disorder characterized by extension of the fibroinflammatory process beyond the thyroid capsule. Due to the nature of this lesion, fine-needle aspiration often yields scant material and may be interpreted as non-diagnostic. In this report, we describe cytologic features that allow the cytopathologist to favor a diagnosis of Riedel thyroiditis, thereby guiding appropriate further work-up and management.

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.

Current preoperative diagnostic procedures for thyroid nodules rely mainly on the cytological interpretation of fine-needle aspirates (FNAs). DNA microarray analysis has been shown to reliably distinguish benign and malignant thyroid nodules in surgically resected specimens, but its diagnostic potential in thyroid FNA has not been examined. In the present study, the expression profiles of 50 benign thyroid lesions and papillary thyroid carcinoma tissue samples were compared, generating a list of 25 differentially expressed genes from this training set. A test set of 22 FNA specimens was evaluated by unsupervised hierarchical cluster analysis using this gene list, and the results were compared to FNA cytology. FNA specimens were found to fall into three clusters: malignant (n = 10), benign (n = 7), and indeterminate (n = 5). The benign and malignant groups showed complete concordance with the final histological diagnosis except for one histologically benign lesion, which was rediagnosed as follicular variant of papillary thyroid carcinoma on histological review. Paired analysis between FNA and matched tissues samples illustrated adequate sampling with FNA. These results illustrate that microarray analysis of FNA is feasible and has the potential to improve the accuracy of FNA in categorizing benign from malignant lesions beyond routine cytological evaluation.

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