The histological spectrum and immunoprofile of head and neck NUT carcinoma: A multicentre series of 30 cases.

BACKGROUND AND AIM: Head and neck nuclear protein of testis carcinoma (HN-NUT) is a rare form of carcinoma diagnosed by NUT immunohistochemistry positivity and/or NUTM1 translocation. Although the prototype of HN-NUT is a primitive undifferentiated round cell tumour (URC) with immunopositivity for squamous markers, it is our observation that it may assume variant histology or immunoprofile. METHODS: We conducted a detailed clinicopathological review of a large retrospective cohort of 30 HN-NUT, aiming to expand its histological and immunohistochemical spectrum. RESULTS: The median age of patients with HN-NUT was 39 years (range = 17-86). It affected the sinonasal tract (43%), major salivary glands (20%), thyroid (13%), oral cavity (7%), larynx (7%), neck (7%) and nasopharynx (3%). Although most cases of HN-NUT (63%) contained a component of primitive URC tumour, 53% showed other histological features and 37% lacked a URC component altogether. Variant histological features included basaloid (33%), differentiated squamous/squamoid (37%), clear cell changes (13%), glandular differentiation (7%) and papillary architecture (10%), which could co-exist. While most HN-NUT were positive for keratins, p63 and p40, occasional cases (5-9%) were entirely negative. Immunopositivity for neuroendocrine markers and thyroid transcription factor-1 was observed in 33 and 36% of cases, respectively. The outcome of HN-NUT was dismal, with a 3-year disease specific survival of 38%. CONCLUSIONS: HN-NUT can affect individuals across a wide age range and arise from various head and neck sites. It exhibits a diverse spectrum of histological features and may be positive for neuroendocrine markers, potentially leading to underdiagnosis. A low threshold to perform NUT-specific tests is necessary to accurately diagnose HN-NUT.

Comprehensive Molecular Characterization of Polymorphous Adenocarcinoma, Cribriform Subtype: Identifying Novel Fusions and Fusion Partners.

Polymorphous adenocarcinoma (PAC) is a common, usually low-grade salivary gland carcinoma. While conventional PACs are most associated with PRKD1 p.E710D hotspot mutations, the cribriform subtype is often associated with gene fusions in PRKD1, PRKD2, or PRKD3. These fusions have been primarily identified by fluorescence in situ hybridization (FISH) analysis, with a minority evaluated by next-generation sequencing (NGS). Many of the reported fusions were detected by break-apart FISH probes and therefore have unknown partners or were negative by FISH altogether. In this study, we aimed to further characterize the fusions associated with PAC with NGS. Fifty-four PACs (exclusively cribriform and mixed/intermediate types to enrich the study for fusion-positive cases) were identified and subjected to NGS. Fifty-one cases were successfully sequenced, 28 of which demonstrated gene fusions involving PRKD1, PRKD2, or PRKD3. There were 10 cases with the PRKD1 p.E710D mutation. We identified a diverse group of fusion partners, including 13 novel partners, 3 of which were recurrent. The most common partners for the PRKD genes were ARID1A and ARID1B. The wide variety of involved genes is unlike in other salivary gland malignancies and warrants a broader strategy of sequencing for molecular confirmation for particularly challenging cases, as our NGS study shows.

A novel low-grade nasopharyngeal adenocarcinoma characterized by a GOLGB1-BRAF fusion gene.

Nasopharyngeal adenocarcinoma is a rare malignancy that is classified into conventional/surface- and salivary-types. Herein we report the case of a 52-year-old male who presented with a right nasopharyngeal mass and right-sided hearing loss. Diagnostic imaging revealed a circumscribed 1.7 cm mass centred in the right antero-lateral aspect of the nasopharynx. A biopsy showed a gland-forming neoplasm that was in continuity with the surface epithelium. The tumor exhibited a nested to micro-papillary architecture, with mild cytologic atypia. Immunohistochemistry demonstrated diffuse staining for CK7, SOX10, and p16; the abluminal layer was highlighted by CK5 and p63, while the luminal cells expressed CD117. The tumor was not amenable to subclassification and was diagnosed as a low-grade nasopharyngeal adenocarcinoma, not otherwise specified (NOS). Subsequent RNA sequencing was performed which identified a novel GOLGB1-BRAF fusion product. Based on its unique morphology and molecular findings, this is presumed to represent a novel subtype of nasopharyngeal adenocarcinoma. In addition to being of diagnostic relevance, this fusion may ultimately represent a potential therapeutic target.

Targeted RNA sequencing in the routine clinical detection of fusion genes in salivary gland tumors.

Salivary gland tumors represent a diverse group of neoplasms that occasionally pose a diagnostic challenge for pathologists, particularly with limited sampling. Gene fusions, which may reflect genetic drivers, are increasingly recognized in a subset of these neoplasms, and can be leveraged for diagnostic purposes. We performed a retrospective analysis on a cohort of 80 benign and malignant salivary gland tumors, enriched for subtypes known to harbor recurrent fusion events, to validate the diagnostic use of a targeted RNA sequencing assay to detect fusion transcripts. Testing identified fusion genes in 71% (24/34) of pleomorphic adenoma and carcinoma-ex-pleomorphic adenoma, with 56% of cases showing rearrangement of PLAG1 and 15% HMGA2. In addition to confirming known partners for these genes, novel PLAG1 fusion partners were identified, including DSTN, NTF3, and MEG3; CNOT2 was identified as a novel fusion partner for HMGA2. In adenoid cystic carcinoma, 95% of cases (19/20) were positive for a fusion event. MYB was rearranged in 60% (12/20), MYBL1 in 30% (6/20), and NFIB in 5% (1/20); two tumors exhibited novel fusion products, including NFIB-TBPL1 and MYBL1-VCPIP1. Fusion genes were identified in 64% (9/14) of cases of mucoepidermoid carcinoma; MAML2 was confirmed to partner with either CRTC1 (43%) or CRTC3 (21%). One salivary duct carcinoma was found to harbor a novel RAPGEF6-ACSL6 fusion gene. Finally, as anticipated, gene fusions were not detected in any of the five acinic cell carcinomas included in the cohort. In summary, targeted RNA sequencing represents a diagnostically useful ancillary technique for identifying a variety of existing, and novel, fusion transcripts in the classification of salivary gland neoplasms.

Subcellular differential expression of Ep-ICD in oral dysplasia and cancer is associated with disease progression and prognosis.

BACKGROUND: Identification of patients with oral dysplasia at high risk of cancer development and oral squamous cell carcinoma (OSCC) at increased risk of disease recurrence will enable rigorous personalized treatment. Regulated intramembranous proteolysis of Epithelial cell adhesion molecule (EpCAM) resulting in release of its intracellular domain Ep-ICD into cytoplasm and nucleus triggers oncogenic signaling. We analyzed the expression of Ep-ICD in oral dysplasia and cancer and determined its clinical significance in disease progression and prognosis. METHODS: In a retrospective study, immunohistochemical analysis of nuclear and cytoplasmic Ep-ICD and EpEx (extracellular domain of EpCAM), was carried out in 115 OSCC, 97 oral dysplasia and 105 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 60 months for oral dysplasia and OSCC patients. Disease-free survival (DFS) was determined by Kaplan-Meier method and multivariate Cox regression analysis. RESULTS: In comparison with normal oral tissues, significant increase in nuclear Ep-ICD and membrane EpEx was observed in dysplasia, and OSCC (p = 0.013 and < 0.001 respectively). Oral dysplasia patients with increased overall Ep-ICD developed cancer in short time period (mean = 47 months; p = 0.044). OSCC patients with increased nuclear Ep-ICD and membrane EpEx had significantly reduced mean DFS of 33.7 months (p = 0.018). CONCLUSIONS: Our study provided clinical evidence for Ep-ICD as a predictor of cancer development in patients with oral dysplasia and recurrence in OSCC patients, suggesting its potential utility in enhanced management of those patients detected to have increased risk of progression to cancer and recurrence in OSCC patients.

Prognostic significance of cytoplasmic S100A2 overexpression in oral cancer patients.

BACKGROUND: Oral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and 5-year survival rates are about 50%. Identification of patients at high risk of recurrence will enable rigorous personalized post-treatment management. Most novel biomarkers have failed translation for clinical use because of their limited successful validation in external patient cohorts. The aim of this study was to determine the prognostic significance of alterations in sub-cellular expression of S100A2, a pro-tumorigenic calcium binding protein, identified as a candidate biomarker in our proteomic analysis in OSCC and validation of its clinical utility in an external cohort. METHODS: In a retrospective study, immunohistochemical analysis of S100A2 was carried out in 235 Indian OSCC (Test set) and 129 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 122 months for OSCC patients following the REMARK criteria. The findings were validated in an external cohort (Validation set 115 Canadian OSCC and 51 normal tissues) and data analyzed using the R package. RESULTS: Significant increase in cytoplasmic and decrease in nuclear S100A2 expression was observed in OSCC in comparison with normal tissues. Cox multivariable regression analysis internally and externally validated cytoplasmic S100A2 association with tumor recurrence. Kaplan Meier analysis of patients stratified to high and low risk groups showed significantly different recurrence free survival (Test set- log rank test, p = 0.005, median survival 16 and 69 months respectively and Validation set - p < 0.00001, median survival 9.4 and 59.9 months respectively); 86% and 81% of patients who had recurrence were correctly stratified into the high risk group. Seventy percent and 81% patients stratified into low risk group did not show cancer recurrence within 1 year in Test and Validation sets. CONCLUSIONS: Our study provided clinical evidence for the potential of cytoplasmic S100A2 overexpression as a predictor of recurrence risk in OSCC patients. A unique translational aspect of our study is validation of S100A2 as prognostic marker in two independent cohorts (Canadian and Indian) suggesting this protein is likely to find widespread utility in clinical practice for identifying oral cancer patients at high risk of disease recurrence.

S100A7 overexpression is a predictive marker for high risk of malignant transformation in oral dysplasia.

Early detection of oral lesions (OLs) at high risk of cancer development is of utmost importance for intervention. There is an urgent unmet clinical need for biomarkers that allow identification of high-risk OLs. Recently, we identified and verified a panel of five candidate protein biomarkers namely S100A7, prothymosin alpha, 14-3-3ζ, 14-3-3σ and heterogeneous nuclear ribonucleoprotein K using proteomics to distinguish OLs with dysplasia and oral cancers from normal oral tissues. The objective of our study was to evaluate the potential of these candidate protein biomarkers for identification of oral dysplastic lesions at high risk of cancer development. Using immunohistochemistry, we analyzed expressions of these five candidate protein biomarkers in 110 patients with biopsy-proven oral dysplasia and known clinical outcome and determined their correlations with p16 expression and HPV 16/18 status. Kaplan-Meier survival analysis showed reduced oral cancer-free survival (OCFS) of 68.6 months (p = 0.007) in patients showing cytoplasmic S100A7 overexpression when compared to patients with weak or no S100A7 immunostaining in cytoplasm (mean OCFS = 122.8 months). Multivariate Cox regression analysis revealed cytoplasmic S100A7 overexpression as the most significant candidate marker associated with cancer development in dysplastic lesions (p = 0.041, hazard ratio = 2.36). In conclusion, our study suggested the potential of S100A7 overexpression in identifying OLs with dysplasia at high risk of cancer development.

Discriminating Interpatient Variabilities of RAS Gene Variants for Precision Detection of Thyroid Cancer.

Importance: Interpatient variabilities in genomic variants may reflect differences in tumor statuses among individuals. Objectives: To delineate interpatient variabilities in RAS variants in thyroid tumors based on the fifth World Health Organization classification of thyroid neoplasms and assess their diagnostic significance in cancer detection among patients with thyroid nodules. Design, Setting, and Participants: This prospective diagnostic study analyzed surgically resected thyroid tumors obtained from February 2016 to April 2022 and residual thyroid fine-needle aspiration (FNA) biopsies obtained from January 2020 to March 2021, at Mount Sinai Hospital, Toronto, Ontario, Canada. Data were analyzed from June 20, 2022, to October 15, 2023. Exposures: Quantitative detection of interpatient disparities of RAS variants (ie, NRAS, HRAS, and KRAS) was performed along with assessment of BRAF V600E and TERT promoter variants (C228T and C250T) by detecting their variant allele fractions (VAFs) using digital polymerase chain reaction assays. Main Outcomes and Measures: Interpatient differences in RAS, BRAF V600E, and TERT promoter variants were analyzed and compared with surgical histopathologic diagnoses. Malignancy rates, sensitivity, specificity, positive predictive values, and negative predictive values were calculated. Results: A total of 438 surgically resected thyroid tumor tissues and 249 thyroid nodule FNA biopsies were obtained from 620 patients (470 [75.8%] female; mean [SD] age, 50.7 [15.9] years). Median (IQR) follow-up for patients who underwent FNA biopsy analysis and subsequent resection was 88 (50-156) days. Of 438 tumors, 89 (20.3%) were identified with the presence of RAS variants, including 51 (11.6%) with NRAS, 29 (6.6%) with HRAS, and 9 (2.1%) with KRAS. The interpatient differences in these variants were discriminated at VAF levels ranging from 0.15% to 51.53%. The mean (SD) VAF of RAS variants exhibited no significant differences among benign nodules (39.2% [11.2%]), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTPs) (25.4% [14.3%]), and malignant neoplasms (33.4% [13.8%]) (P = .28), although their distribution was found in 41.7% of NIFTPs and 50.7% of invasive encapsulated follicular variant papillary thyroid carcinomas (P < .001). RAS variants alone, regardless of a low or high VAF, were significantly associated with neoplasms at low risk of tumor recurrence (60.7% of RAS variants vs 26.9% of samples negative for RAS variants; P < .001). Compared with the sensitivity of 54.2% (95% CI, 48.8%-59.4%) and specificity of 100% (95% CI, 94.8%-100%) for BRAF V600E and TERT promoter variant assays, the inclusion of RAS variants into BRAF and TERT promoter variant assays improved sensitivity to 70.5% (95% CI, 65.4%-75.2%), albeit with a reduction in specificity to 88.8% (95% CI, 79.8%-94.1%) in distinguishing malignant neoplasms from benign and NIFTP tumors. Furthermore, interpatient differences in 5 gene variants (NRAS, HRAS, KRAS, BRAF, and TERT) were discriminated in 54 of 126 indeterminate FNAs (42.9%) and 18 of 76 nondiagnostic FNAs (23.7%), and all tumors with follow-up surgical pathology confirmed malignancy. Conclusions and Relevance: This diagnostic study delineated interpatient differences in RAS variants present in thyroid tumors with a variety of histopathological diagnoses. Discrimination of interpatient variabilities in RAS in combination with BRAF V600E and TERT promoter variants could facilitate cytology examinations in preoperative precision malignancy diagnosis among patients with thyroid nodules.

SMARCB1 (INI1)-deficient sinonasal carcinoma manifesting as oral lesions: A report of two cases.

BACKGROUND: Sinonasal carcinomas represent a rare group of malignancies, accounting for less than 5% of all head and neck cancers and a worldwide incidence of less than 1 case per 100 000 inhabitants annually. Despite the restricted anatomical location, sinonasal carcinomas harbor some of the most histologically and molecularly diverse groups of tumors. SMARCB1 (INI1)-deficient sinonasal carcinomas are locally aggressive tumors commonly detected late, leading to devastating morbidity and mortality. CASE REPORT: We present two cases of SMARCB1-deficient sinonasal carcinoma involving the oral cavity and presenting as progressive radiolucent lesions with local swelling associated with maxillary dentition and alveolar bone. Both cases were initially considered odontogenic in origin and involved the destruction of the left anterior maxilla. CONCLUSION: Given the rarity and the variable presentation of these tumors, they pose a challenge for head and neck surgeons, dentists, and pathologists due to the potential overlapping features with odontogenic and non-odontogenic inflammatory and neoplastic lesions. These cases highlight the importance of a multidisciplinary team and include SMARCB1-deficient sinonasal carcinomas in the differential diagnosis of destructive lesions of the maxilla.

Secretome proteins as candidate biomarkers for aggressive thyroid carcinomas.

Using proteomics in tandem with bioinformatics, the secretomes of nonaggressive and aggressive thyroid carcinoma (TC) cell lines were analyzed to detect potential biomarkers for tumor aggressiveness. A panel of nine proteins, activated leukocyte cell adhesion molecule (ALCAM/CD166), tyrosine-protein kinase receptor (AXL), amyloid beta A4 protein, amyloid-like protein 2, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase isozyme M2, phosphatase 2A inhibitor (SET), and protein kinase C inhibitor protein 1 (14-3-3 zeta) was chosen to confirm their expression in TC patients' sera and tissues. Increased presurgical circulating levels of ALCAM were associated with aggressive tumors (p = 0.04) and presence of lymph node metastasis (p = 0.018). Increased serum AXL levels were associated with extrathyroidal extension (p = 0.027). Furthermore, differential expression of amyloid beta A4 protein, AXL, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase muscle isozyme M2, and SET was observed in TC tissues compared to benign nodules. Decreased nuclear expression of AXL can detect malignancy with 90% specificity and 100% sensitivity (AUC = 0.995, p < 0.001). In conclusion, some of these proteins show potential for future development as serum and/or tissue-based biomarkers for TC and warrant further investigation in a large cohort of patients.

Cribriform adenocarcinoma of the minor salivary glands: a case report.

BACKGROUND: Polymorphous adenocarcinoma is the third most common malignant salivary gland tumor. Within polymorphous adenocarcinoma, cribriform adenocarcinoma of salivary glands is a rare subtype and resembles papillary thyroid carcinoma histopathologically. Diagnostically, cribriform adenocarcinoma of salivary glands is challenging for pathologists and surgeons alike as initial presentation and cytologic nuclear features can be easily confused with papillary thyroid carcinoma arising from a thyroglossal duct remnant or lingual thyroid. CASE PRESENTATION: A healthy 64-year-old Caucasian woman presented to a community otolaryngologist with a 4-year history of progressive postnasal drip, globus sensation, and eventual dysphonia. Flexible fiberoptic laryngoscopy showed a large, smooth, vallecular lesion filling the oropharynx. Computed tomography imaging of the neck showed a rounded heterogeneous mass centered within the right aspect of the oropharynx measuring 4.2 × 4.4 × 4.5 cm. Fine needle aspiration biopsy was suspicious for papillary carcinoma due to microscopic findings of malignant cells, nuclear grooves, and a powdery chromatin pattern. In the operating room, the tumor was resected en bloc using a lateral pharyngotomy approach with partial resection of the right lateral hyoid. A limited cervical lymphadenectomy was performed to facilitate the lateral pharyngotomy approach and two out of three lymph nodes demonstrated regional metastatic disease. Nuclear grooves, nuclear membrane notching, and occasional intranuclear pseudoinclusions were identified, which are overlapping histopathological characteristics of papillary thyroid carcinoma and cribriform adenocarcinoma of salivary glands. It was negative for thyroglobulin and thyroid transcription factor-1, which was in keeping with cribriform adenocarcinoma of salivary glands rather than papillary thyroid carcinoma. CONCLUSION: It is difficult to distinguish cribriform adenocarcinoma of salivary glands from papillary thyroid carcinoma solely by cytology, and the distinct characteristics of regional lymph node metastasis coupled with nuanced histologic differences should be emphasized in the evaluation of patients presenting with neck lymphadenopathy and an unknown primary or tongue mass. If sufficient fine needle aspiration biopsy material is available, thyroid transcription factor-1, thyroglobulin, or molecular testing may prove useful in differentiating cribriform adenocarcinoma of salivary glands from papillary thyroid carcinoma. A misdiagnosis of papillary thyroid carcinoma may lead to inappropriate treatment including unnecessary thyroidectomy. Therefore, it is critical for both pathologists and surgeons to be aware of this uncommon entity to avoid misdiagnosis and subsequent mismanagement.

A sinonasal NUT midline carcinoma in an 84-year-old man undergoing radiation and proton therapy.

NUT midline carcinomas are rare, aggressive, and poorly differentiated tumors that must be considered in the differential diagnosis of midline head and neck tumors. Despite the scarce data, proton therapy could be an option for some patients.

Facilitation of Definitive Cancer Diagnosis With Quantitative Molecular Assays of BRAF V600E and TERT Promoter Variants in Patients With Thyroid Nodules.

Importance: Molecular testing of the presence of pathogenic genomic variants in a tumor without quantifying the variant allele fraction (VAF) does not differentiate the variation extent among tumors, often resulting in an inconclusive diagnosis because of interpatient variability. Objective: To examine the association between the quantification of VAFs of BRAF V600E and TERT promoter variants and a definitive cancer diagnosis of thyroid tumors. Design, Setting, and Participants: This diagnostic study analyzed a cohort of 378 surgically resected thyroid tumors with a maximum dimension of 1 cm or larger between March 15, 2016, and March 16, 2020, and a separate cohort of 217 residual thyroid fine-needle aspiration (FNA) biopsy specimens obtained from January 22, 2020, to March 2, 2021, at Mount Sinai Hospital, Toronto, Ontario, Canada. Data analysis was conducted between February 1, 2021, and February 1, 2023. Exposures: Quantitative VAF assays of BRAF V600E and TERT promoter variants (C228T and C250T) were performed by digital polymerase chain reaction molecular assays. Main Outcomes and Measures: The VAFs of BRAF V600E and TERT promoter variants were correlated with tumor histologic diagnoses and histopathologic features to delineate the association of VAF assays with tumor malignancy. The receiver operating characteristic curve analysis, sensitivity, specificity, positive predictive value, negative predictive value, and logistic regression analysis based on follow-up histopathologic types were used to determine the diagnostic utility of the quantitative molecular assays. Results: A total of 595 specimens, including 378 surgically resected thyroid tumors and 217 thyroid nodule FNA biopsy specimens, were collected from 580 patients (436 [75.2%] female with a mean [SD] age of 50 [16] years and 144 [24.8%] male with a mean [SD] age of 55 [14] years). Sensitive VAF assays of 378 thyroid tumors revealed the presence of the BRAF V600E variant in 162 tumors (42.9%), with 26 (16.0%) at a low VAF of 1% or less and 136 (84.0%) at a high VAF of greater than 1%, and the presence of TERT promoter variants in 49 tumors (13.0%), including 45 C228T variants (91.8%), 15 (33.3%) of which were quantified as having a low VAF (≤1%) and 30 (66.7%) as having a high VAF (>1%), and 4 C250T variants (8.2%) with VAFs between 40.0% and 47.0%. All tumors detected with BRAF V600E and/or TERT promoter variants, whether at low or high VAFs, received a definitive cancer diagnosis. Further analysis delineated a significant association between high VAFs of either variant individually or different VAF levels for both variants in coexistence and aggressive histopathologic features of tumors. Excluding low VAFs assisted in identifying patients at an intermediate-to-high risk of recurrence (odds ratio, 5.3; 95% CI, 1.9-14.6; P = .001). The VAF assays on the residual FNA biopsy specimens showed a high agreement to those on surgical tissues (κ = 0.793, P < .001) and stratified malignancy in 40 of 183 indeterminate FNA cases (21.9%), with a sensitivity of 93.8% (95% CI, 67.7%-99.7%), specificity of 90.0% (95% CI, 75.4%-96.7%), positive predictive value of 78.9% (95% CI, 53.9%-93.0%), and negative predictive value of 97.3% (95% CI, 84.2%-99.9%). Conclusions and Relevance: This diagnostic study suggests that sensitive quantitative VAF assays of BRAF V600E and TERT promoter variants can elucidate the interpatient variability in tumors and facilitate a definitive cancer diagnosis of thyroid nodules by differentiating the variation extent of genomic variants, even at low VAFs.

A 10-Year Review of Intraoral Salivary Gland Tumor Diagnoses: Diagnostic Challenges and Inter-Observer Agreement.

BACKGROUND: Salivary gland tumors (SGT) are a diverse group of neoplasms arising from the major and minor glands. The oral cavity is the most common site for minor SGT (IMSGT), and these lesions frequently pose a challenge to the pathologist due to overlapping histopathological features and limited material for analysis. Our objective was to determine specific clinical and histopathological features associated with challenges in IMSGT diagnoses and pathologists' agreement. METHODS: We conducted a retrospective analysis of 248 IMSGT received between 2010 and 2019. We evaluated the diagnostic challenge of the cases by stratifying according to whether a definitive, favored, or indeterminate (challenging) diagnosis was provided. Inter-observer agreement and concordance of biopsy diagnoses with the final diagnoses after tumor resection were evaluated. RESULTS: Of the 248 biopsies, 191 had a definitive diagnosis, 38 favored diagnoses, and 19 were indeterminate. The predominant diagnoses considered for the indeterminate category were pleomorphic adenoma/myoepithelioma (PA), polymorphous adenocarcinoma (PAC), adenoid cystic carcinoma (AdCC), and low-grade adenocarcinoma. Using multivariate analysis of clinical features, younger patient age, smaller tumor size, and larger biopsy size increased the likelihood of a definitive diagnosis (p = 0.014, p = 0.037, p = 0.012). The inter-observer agreement for 68 representative cases was moderate overall (Fleiss's Kappa 0.575) and good for the 40 cases with a definitive diagnosis (Fleiss's Kappa 0.66). Sixty-five biopsy diagnoses were matched with corresponding tumor resection diagnoses and found to show a good concordance (Cramer's V test 0.76). The discordant diagnoses predominantly involved PA, carcinoma exPA, PAC, AdCC, and adenocarcinoma NOS. CONCLUSION: Diagnostic challenges in IMSGT incisional biopsies were infrequent, especially if multiple pathologists were consulted. PA, PAC, AdCC, and adenocarcinoma NOS were the histologic types more commonly posing diagnostic challenges. Younger patient age, smaller tumor size, and larger biopsy are associated with a definitive diagnosis. This data highlights the importance of appropriate sampling in IMSGT.

Immunohistochemical analysis based Ep-ICD subcellular localization index (ESLI) is a novel marker for metastatic papillary thyroid microcarcinoma.

BACKGROUND: Thyroid cancer is among the fastest growing malignancies; almost fifty-percent of these rapidly increasing incidence tumors are less than or equal to 1cm in size, termed papillary thyroid microcarcinoma (PTMC). The management of PTMC remains a controversy due to differing natural history of these patients. Epithelial cell adhesion molecule (EpCAM) is comprised of an extracellular domain (EpEx), a single transmembrane domain and an intracellular domain (Ep-ICD). Our group reported nuclear Ep-ICD correlated with poor prognosis in thyroid cancer (Ralhan et al., BMC Cancer 2010,10:331). Here in, we hypothesized nuclear and cytoplasmic accumulation of Ep-ICD and loss of membranous EpEx may aid in distinguishing metastatic from non-metastatic PTMC, which is an important current clinical challenge. To test our hypothesis, Ep-ICD and EpEx expression levels were analyzed in PTMC and the staining was correlated with metastatic potential of these carcinomas. METHODS: Thirty-six PTMC patients (tumor size 0.5 - 1cm; metastatic 8 cases and non-metastatic 28 cases) who underwent total thyroidectomy were selected. The metastatic group consisted of patients who developed lymph node or distant metastasis at diagnosis or during follow up. The patients' tissues were stained for Ep-ICD and EpEx using domain specific antibodies by immunohistochemistry and evaluated. RESULTS: PTMC patients with metastasis had higher scores for nuclear and cytoplasmic Ep-ICD immunostaining than the patients without metastasis (1.96 ± 0.86 vs. 1.22 ± 0.45; p = 0.007 and 5.37 ± 0.33 vs. 4.72 ± 1.07; p = 0.016, respectively). Concomitantly, the former had lower scores for membrane EpEx than the non-metastatic group (4.64 ± 1.08 vs. 5.64 ± 1.51; p = 0.026). An index of aggressiveness, Ep-ICD subcellular localization index (ESLI), was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep - ICDnuc + Ep - ICDcyt + loss of membranous EpEx]. Notably, ESLI correlated significantly with lymph node metastasis in PTMC (p = 0.008). CONCLUSION: Nuclear and cytoplasmic Ep-ICD expression and loss of membranous EpEx were found to correlate positively with metastasis in PTMC patients. In addition, ESLI had the potential to identify metastatic behavior in PTMC which could serve as a valuable tool for solving a current dilemma in clinical practice.

Development of a Molecular Assay for Detection and Quantification of the BRAF Variation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens.

Importance: Thyroid cancer, predominantly papillary thyroid carcinoma (PTC), is common, but an estimated 30% of ultrasonography-guided fine-needle aspiration (FNA) biopsies of thyroid nodules are indeterminate. BRAF variation, associated with poor clinicopathological characteristics, is a useful molecular marker for diagnostics. Objective: To develop a sensitive molecular assay for BRAF V600E detection in remaining tissue of thyroid FNA biopsies to identify patients with cancer carrying a BRAF variation. Design, Setting, and Participants: This diagnostic study used tumor tissue from surgical formalin-fixed, paraffin-embedded (FFPE) specimens and residual tissue from thyroid FNA biopsies for genomic DNA extraction. FFPE specimens served as the validation set, and residual tissue from FNA biopsies served as the test set. A molecular assay was developed for accurate detection of BRAF V600E variation using locked nucleic acid (LNA) probe-based droplet digital polymerase chain reaction (dPCR), and the assay was validated by BRAF V600E immunohistochemical staining (IHC). The study was conducted between February 2019 and May 2021. Results: A total of 271 specimens, including 77 FFPE specimens (with a follow-up of 48 matched surgical specimens) and 146 residual FNA samples, were collected from 223 patients (mean [SD] age, 53.8 [15.3] years; 174 [78.0%] women; 49 [22.0%] men). The molecular assay by dPCR was first established to specifically and accurately detect and quantify wild-type BRAF and variant BRAF in DNA from human follicular thyroid carcinoma-derived FTC-133 and papillary thyroid carcinoma-derived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x; R2 = 0.9996) with sensitivity at 0.02 copies/µL and reproducibility in detecting variant DNA at various dilutions(coefficient of variance in 0.3% DNA, 9.63%; coefficient of variance in 1.0% DNA, 7.41%). In validation testing, the dPCR assay and IHC staining exhibited 100% specificity in concordantly identifying BRAF V600E in PTCs (κ = 0.873; P < .001) and sensitivity of 32.0% (95% CI, 19.1% to 44.9%) in dPCR and 26.0% (95% CI, 13.1% to 38.9%) in IHC staining, with an improvement by 23.08% in dPCR compared with the IHC staining. The dPCR assay further detected BRAF V600E in 39 of 146 residual FNA specimens (26.7%). At short-term follow-up, 48 patients, including 14 of 39 patients with BRAF variation and 34 of 107 patients without BRAF variation on residual FNA specimens, underwent resection. The dPCR assay of BRAF status in the matched surgical specimens showed BRAF V600E variations in 12 patients and wild-type BRAF in 36 patients, with a high agreement to that in residual tissue of FNA specimens (κ = 0.789; P < .001). Among 14 patients with BRAF variations on residual FNA, 13 were diagnosed with PTC and 1 was diagnosed with anaplastic thyroid cancer at the thyroidectomy. Conclusions and Relevance: This diagnostic study developed a sensitive molecular assay for detection and quantification of BRAF V600E variation in residual tissue from thyroid FNA biopsies to identify patients with cancer harboring BRAF V600E in a cost-effective manner, highlighting the clinical value of molecular assay of the remaining FNA tissue in the management of thyroid nodules.

A Poorly Differentiated Non-keratinizing Sinonasal Squamous Cell Carcinoma with a Novel ETV6-TNFRSF8 Fusion Gene.

Squamous cell carcinoma of the sinonasal tract is relatively rare and morphologically and genetically heterogeneous. We report the case of an adult male with a left sphenoid sinus mass. A biopsy revealed an undifferentiated carcinoma composed of sheets of epithelioid cells lacking keratinization and glandular formation. The tumor was associated with a prominent lymphoplasmacytic inflammatory infiltrate. Immunohistochemical staining demonstrated diffuse expression of pankeratin and p63; it was negative for p16. In addition, EBER was also negative. Morphologically the findings raised the possibility of non-keratinizing squamous cell carcinoma. RNA sequencing was undertaken to exclude the possibility of NUT carcinoma; interestingly, this revealed a novel ETV6-TNFRSF8 fusion transcript, which was independently confirmed by fluorescence in situ hybridization. The current case is illustrative because it broadens our understanding of the molecular pathogenesis of non-keratinizing squamous cell carcinoma and adds to the diversity of ETV6-rearranged malignancies.

DEK-AFF2 Carcinoma of the Sinonasal Region and Skull Base: Detailed Clinicopathologic Characterization of a Distinctive Entity.

A novel DEK-AFF2 fusion was recently reported in 4 nonkeratinizing squamous cell carcinomas of the sinonasal region and skull base, including 1 with exceptional response to immunotherapy, but it is not yet clear if this rearrangement defines a unique clinicopathologic category or represents a rare event. This study aims to characterize a larger cohort of carcinomas with DEK-AFF2 fusions to assess whether they truly constitute a distinctive entity. Among 27 sinonasal and skull base nonkeratinizing squamous cell carcinoma that were negative for human papillomavirus and Epstein-Barr virus, RNA sequencing identified DEK-AFF2 fusions in 13 cases (48%). Nine were centered in the nasal cavity, 2 in the middle ear/temporal bone, 1 in the nasopharynx, and 1 in the orbit. These tumors displayed recurrent histologic features including (1) complex endophytic and exophytic, frequently papilloma-like growth, (2) transitional epithelium with eosinophilic to amphophilic cytoplasm, (3) absent or minimal keratinization with occasional compact keratin pearls, (4) monotonous nuclei, and (5) prominent tumor-infiltrating neutrophils or stromal lymphocytes. This appearance not only overlaps with high-grade basaloid sinonasal carcinomas but also with benign papillomas and tumors reported as low-grade papillary Schneiderian carcinoma. However, DEK-AFF2 carcinomas showed frequent local recurrence, cervical lymph node metastases, and distant metastasis with 2 deaths from disease, confirming they are aggressive malignancies despite relatively bland histology. Overall, the distinctive molecular, histologic, and clinical features of DEK-AFF2 carcinomas suggest they represent a unique entity in the sinonasal region. This tumor merits increased pathologic recognition to better understand its prognostic and therapeutic implications.

Secretome-based identification and characterization of potential biomarkers in thyroid cancer.

In search of thyroid cancer biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of 46 high-confidence identifications, 6 proteins were considered for verification in thyroid cancer patients' tissue and blood. The localization of two proteins, nucleolin and prothymosin-α (PTMA), was confirmed in TPC-1 and CAL62 cells by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells in NOD/SCID/γ mice and human thyroid cancers (48 tissues). Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic compared to papillary and poorly differentiated carcinomas. Nuclear expression of nucleolin was observed in all subtypes of thyroid carcinomas, along with faint cytoplasmic expression in anaplastic cancers. Importantly, PTMA, nucleolin, clusterin, cysteine-rich angiogenic inducer 61, enolase 1, and biotinidase were detected in thyroid cancer patients' sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. In conclusion, we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify novel proteins that can be independently verified in cell lines, xenografts, tumor tissues, and blood samples of thyroid cancer patients. These observations support their potential utility as minimally invasive biomarkers for thyroid carcinomas and their application in management of these diseases upon future validation.

EpCAM nuclear localization identifies aggressive thyroid cancer and is a marker for poor prognosis.

BACKGROUND: Proteolytic cleavage of the extracellular domain (EpEx) of Epithelial cell adhesion molecule (EpCAM) and nuclear signaling by its intracellular oncogenic domain Ep-ICD has recently been implicated in increased proliferation of cancer cells. The clinical significance of Ep-ICD in human tumors remains an enigma. METHODS: EpEx, Ep-ICD and beta-catenin immunohistochemistry using specific antibodies was conducted on 58 archived thyroid cancer (TC) tissue blocks from 34 patients and correlated with survival analysis of these patients for up to 17 years. RESULTS: The anaplastic (ATC) and aggressive thyroid cancers showed loss of EpEx and increased nuclear and cytoplasmic accumulation of Ep-ICD. In contrast, the low grade papillary thyroid cancers (PTC) showed membranous EpEx and no detectable nuclear Ep-ICD. The ATC also showed concomitant nuclear expression of Ep-ICD and beta-catenin. Kaplan-Meier Survival analysis revealed reduced overall survival (OS) for TC patients showing nuclear Ep-ICD expression or loss of membranous EpEx (p < 0.0004), median OS = 5 months as compared to 198 months for patients who did not show nuclear Ep-ICD or demonstrated only membranous EpE. CONCLUSION: We report reciprocal loss of membrane EpEx but increased nuclear and cytoplasmic accumulation of Ep-ICD in aggressive TC; nuclear Ep-ICD correlated with poor OS of TC patients. Thus nuclear Ep-ICD localization may serve as a useful biomarker for aggressive TC and may represent a novel diagnostic, prognostic and therapeutic target for aggressive TC.