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Poorly-differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are associated with a rapidly progressive course and poor median survival. Recently we published promising results in a murine model showing efficient clearance of metastatic tumor burden of human ATC cell line 8505c and an anaplastic patient-derived cell line using healthy donor T cells transduced with a third-generation chimeric antigen receptor (CAR) construct targeting intercellular adhesion molecule-1 (ICAM1). However, patient-derived T cells using an identical ICAM1-targeted CAR construct demonstrated slower tumor-killing in vitro. To investigate the role of immune checkpoint proteins PD-L1 and PD-1 in thyroid cancer and as potential inhibitors of ICAM1-CAR T, we stained formalin-fixed paraffin-embedded archived tissue samples for PD-L1 (papillary thyroid cancer, PTC, n = 17; ATC, n = 13) using immunohistochemistry (IHC) and extracted peripheral blood mononuclear cells (PBMCs) from patient blood samples and healthy donors to evaluate PD-1 expression on CD3+/CD8+ T cells using flow cytometry (healthy donor, n = 2; PTC, n = 7; PDTC/ATC, n = 6). Twelve percent of PTC samples were PD-L1+ (threshold >1% positivity) versus 69% of PDTC/ATC samples (Chi-square, p = 0.012); PD-L1 positivity was associated with circumferential ICAM1 staining on IHC (Chi-square, p = 0.003). Similarly, median percentage of PD-1 expression on PBMC's was higher in PDTC/ATC than PTC (58.1% versus 5.2%, Wilcoxon rank sum test; p = 0.03). Interestingly, expression of elevated PD-1 was seen in PBMC samples from patients in the absence of PD-L1 positivity of the primary tumor (IHC) in 4 of 5 matched tumor samples. This suggests that PD-1 in circulating CD8+ cells may be an important predictor of response to checkpoint inhibitor therapy. Finally, effector:target assays with patient-derived ICAM1-CAR T effector cells (baseline PD-1 expression 96%) against 8505c (PD-L1 100%/ICAM1 68%) and matched patient-derived cell line (PD-L1 >50%/ICAM1 88%) showed enhanced tumor killing in the presence of anti-PD-1, which approached the level of tumor cell death seen with healthy donor ICAM1-CAR T. Our results show that PD-1 is increased in the peripheral blood of patients with poorly differentiated and anaplastic thyroid cancer and is a potential therapeutic target. Furthermore, anti-PD-1 may be used to counter PD-L1+ tumor-induced T cell suppression (or exhaustion) to increase the efficacy of CAR T cells in ATC. Citation Format: Katherine D. Gray, Yogindra Vedvyas, Olivia Kalloo, Enda Shevlin, Theresa Scognamiglio, Marjan Zaman, Andrew Tassler, Moonsoo M. Jin, Rasa Zarnegar, Thomas J. Fahey, Irene M. Min. PD-L1/PD-1 checkpoint inhibition in anaplastic thyroid cancer and enhancement of ICAM1-targeted chimeric antigen receptor (CAR)-T cell tumor lysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2738.

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.

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.

Introduction: Poorly differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet inherently lethal malignancies with limited treatment options. Many malignant tumors, including papillary thyroid cancer (PTC) and ATC, are associated with increased expression of intercellular adhesion molecule-1 (ICAM-1), providing a rationale for utilizing ICAM-1-targeting agents for the treatment of aggressive types of thyroid cancer. Therefore, we developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 to leverage adoptive T cell therapy as a new treatment modality against advanced thyroid cancer. Methods: We created a firefly luciferase-expressing human ATC model in mice that develops systemic metastases very rapidly. ATC engrafted mice were treated with human peripheral blood T cells modified with a lentivirus encoding an ICAM-1 specific CAR (ICAM-1-CAR) to investigate their therapeutic efficacy. Tumor burden was longitudinally measured by whole body bioluminescence imaging of luciferase-positive tumor cells. Effector:target assays consisting of ICAM-1-CAR T cells co-cultured with multiple malignant and non-malignant target cells were used to investigate specific target cell death and ‘off-tumor’ toxicity in vitro using luminescence and flow cytometry. Results: ICAM-1-CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro. Strikingly, despite heterogeneous expression of ICAM-1 in ATC cell lines, addition of cytotoxic CAR T cells induced increased ICAM-1 expression by T cell-derived interferon gamma such that all cell lines became targetable by ICAM-1-CAR T cells. Patient-derived, poorly differentiated PTC cells overexpressed ICAM-1 and were also mostly eliminated by autologous ICAM-1 CAR T cells in vitro. In mice with systemic ATC, a single administration of ICAM-1-CAR-T cells at a clinical dose mediated significant tumor killing with a 100-fold reduction in primary tumor burden compared to pre-treatment. Reductions in tumor burden persisted for over 80 days and treated mice demonstrated significantly improved survival without toxicity. Conclusion: Our findings are the first demonstration of the potential for CAR-T cell therapy for metastatic, advanced thyroid cancers. ICAM-1-CAR T cells demonstrated significant therapeutic efficacy in vitro and in vivo and extended survival benefits in animal models. Citation Format: Irene M. Min, Yogindra Vedvyas, Enda Shevlin, Marjan Zaman, Brian Wyrwas, Weibin Wang, Susan Park, Maureen Moore, Theresa Scognamiglio, Rasa Zarnegar, Thomas J. Fahey, Moonsoo M. Jin. CAR T cells targeting ICAM-1 trigger strong antitumor effects against advanced human thyroid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2017-3624

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 cells of origin of oral cavity squamous cell carcinoma (OCSCC) are unknown. We used a cell lineage tracing approach (adult K14-CreER(TAM); ROSA26 mice transiently treated with tamoxifen) to identify and track normal epithelial stem cells (SCs) in mouse tongues by X-gal staining and to determine if these cells become neoplastically transformed by treatment with a carcinogen, 4-nitroquinoline 1-oxide (4-NQO). Here, we show that in normal tongue epithelia, X-gal(+) cells formed thin columns throughout the entire epithelium 12 weeks after tamoxifen treatment, indicating that the basal layer contains long-lived SCs that produce progeny by asymmetric division to maintain homeostasis. Carcinogen treatment results in a ~10-fold reduction in the total number of X-gal(+) clonal cell populations and horizontal expansion of X-gal(+) clonal cell columns, a pattern consistent with symmetric division of some SCs. Finally, X-gal(+) SCs are present in papillomas and invasive OCSCCs, and these long-lived X-gal(+) SCs are the cells of origin of these tumors. Moreover, the resulting 4-NQO-induced tumors are multiclonal. These findings provide insights into the identity of the initiating cells of oral cancer.

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.

Clinical specimens cultured on two selective fungal media, inhibitory mold agar (IMA) and Sabouraud dextrose agar (SDA), were compared with respect to recovery of fungi. Of the 840 fungal isolates recovered, 69.3% grew on both IMA and SDA; 24.9% grew only on IMA; and 5.8% grew only on SDA, showing that IMA is superior ( P = 0.003).

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.

BACKGROUND: A subset of follicular lesions of the thyroid is encapsulated similar to follicular adenomas but with partial nuclear features suggestive of papillary thyroid carcinoma (PTC), raising the possibility of biologically borderline tumors. METHODS: Gene expression profiling and advanced significance analyses were performed on 50 histologically unequivocal benign and malignant tumors, and a list of 61 differentially expressed genes was generated. By using this 61-gene list, unsupervised hierarchical and K-means cluster analyses were performed on 40 additional tumors, including 15 histologically borderline tumors, 11 benign tumors, and 14 PTCs. RESULTS: Analysis revealed 3 distinct tumor groups—benign, malignant, and intermediate. Tumors in the intermediate group (n = 15) were mostly histologic borderline tumors and had an expression profile overlapping with the benign and malignant groups. Twenty-seven genes were expressed differentially between the benign and intermediate groups, including the cyclic AMP response element-binding protein/p300-interactivator with glutamic acid/aspartic acid-rich carboxy-terminal domain 1 or CITED1 gene and the fibroblast growth factor receptor 2 or FGFR2 gene. Fourteen genes were expressed differentially between the intermediate group and malignant tumors, notably overexpression of the met proto-oncogene and of the high-mobility group adenine/thymine-hook 2 or HMGA2 gene in malignancies. Mutations of the v-raf murine sarcoma viral oncogene homolog B1 or BRAF gene were identified in 4 of 14 malignant tumors but not in benign or intermediate tumors. Patients who had either histologically or molecularly borderline tumors did not have metastasis or recurrences. CONCLUSIONS: Gene expression profiling supported the finding that encapsulated thyroid follicular lesions with partial nuclear features of PTC are biologically borderline tumors that are distinct molecularly from benign and malignant tumors. Cancer 2009. © 2009 American Cancer Society.

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.

Carcinoma ex pleomorphic adenoma (CXPA) is a rare epithelial malignancy that arises from a primary or recurrent pleomorphic adenoma (PA). It may be noninvasive (NI) or invasive. NI CXPA is extremely rare. Preoperative diagnosis on fine needle aspiration (FNA) of CXPA may be difficult and poses a diagnostic challenge to clinicians and pathologists. Herein, we describe the FNA findings of a case of NI-CXPA. A 69-year-old woman presented with rapid enlargement of a stable parotid mass of 25 years. Cytologically, malignant cells were focally associated with metachromatic fibromyxoid matrix that was homogeneous and dense with a vague fibrillary quality. There were cell groups, papillary-like clusters and single malignant cells. The nuclei were pleomorphic with irregularly dispersed chromatin, and the cytoplasm was ill-defined and granular. Nucleoli were small to inconspicuous. Mitoses and necrosis were not seen. Cytological features were not specific for any type of salivary gland carcinoma. The FNA diagnosis was primary high-grade adenocarcinoma of the parotid gland, not otherwise specified. Facial nerve-sparing total parotidectomy was performed, which histologically showed PA interspersed with ducts and nests composed of pleomorphic atypical nuclei surrounded by extensive hyalinization. Single cells were also noted. No capsular infiltration was seen in the entirely sampled tumor. Immunohistochemistry for Ki-67 showed a higher proliferation rate in the malignant ducts and p63 positive cells focally surrounded some of the malignant ducts. Histological diagnosis was NI-CXPA. Accurate diagnosis is important for proper surgical management; however, the preoperative diagnosis of NI-CXPA is difficult to make on FNA.

The cells of origin of oral cavity squamous cell carcinoma (OCSCC) are unknown. We used a cell lineage tracing approach (adult K14-CreERTAM; ROSA 26 mice transiently treated with tamoxifen) to identify and track normal epithelial stem cells (SCs) in mouse tongues by X-gal staining and to determine if these cells become neoplastically transformed by treatment with a carcinogen, 4-nitroquinoline 1-oxide (4-NQO). Here we show that in normal tongue epithelia, X-gal(+) cells formed thin columns throughout the entire epithelium 12 weeks after tamoxifen treatment, indicating that the basal layer contains long lived SCs that produce progeny by asymmetric division to maintain homeostasis. Carcinogen treatment results in a ∼ 10 fold reduction in the total number of X-gal(+) clonal cell populations and horizontal expansion of X-gal(+) clonal cell columns, a pattern consistent with symmetric division of some SCs. Finally, X-gal(+) SCs are present in papillomas and invasive OCSCCs, and these long-lived X-gal(+) SCs are the cells of origin of these tumors. Moreover, the resulting 4-NQO induced tumors are multi-clonal. These findings provide insights into the identity of the initiating cells of oral cancer. Citation Format: Xiao-Han Tang, Theresa Scognamiglio, Lorraine Gudas. Basal stem cells contribute to squamous cell carcinomas in the oral cavity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2014-3036

The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and RARβ2, by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase (Dnmt) 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, respectively), 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 performed 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. While all drug treatments limited the increase in the percentage of PCNA 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3462.