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.

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.

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 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.

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 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.

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.

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.

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.

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.

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.

Microsecretory Adenocarcinoma: A Novel Salivary Gland Tumor Characterized by a Recurrent MEF2C-SS18 Fusion.

Salivary gland adenocarcinoma not otherwise specified (NOS) is a heterogenous group, likely containing distinct tumors not yet characterized. A growing number of low to intermediate-grade salivary carcinomas are now known to harbor tumor-specific gene fusions. On occasion, identifying a novel fusion allows for recognition of a new salivary tumor type, in addition to representing a potential diagnostic tool. We sought to characterize a distinctive salivary gland adenocarcinoma that would previously have been regarded as adenocarcinoma NOS. On the basis of the recognition of 5 morphologically identical, distinct low-grade salivary adenocarcinomas, we used targeted RNA sequencing (RNA-Seq) to determine whether these could be differentiated from other fusion-associated salivary gland tumors. RNA-Seq was performed on all 5 low-intermediate grade adenocarcinomas NOS with near-identical histologic appearances, as well as 23 low-intermediate grade control adenocarcinoma NOS cases that did not resemble the index cases. All 5 index cases harbored a novel MEF2C-SS18 gene fusion, which was independently confirmed by reverse transcriptase-polymerase chain reaction. The MEF2C-SS18-positive cases arose in the oral cavity (4/5) and parotid gland (1/5) of 3 women and 2 men ranging from 21 to 80 years (mean: 46) and shared near-identical histologic features: intercalated duct-like cells with eosinophilic to clear cytoplasm and small, uniform oval nuclei, infiltrative microcysts and cords, abundant intraluminal secretions, and cellular fibromyxoid stroma. Mitotic rates were low; necrosis was absent. All MEF2C-SS18-positive tumors were positive for S100 and p63 and negative for p40, smooth muscle actin, calponin, and mammaglobin. One of the 23 control cases, a parotid tumor, was found to contain a SS18-ZBTB7A gene fusion; it demonstrated similar, but not identical histologic and immunophenotypic features compared with the MEF2C-SS18 cases. The remaining control cases were negative for SS18 and MEF2C rearrangements. A novel MEF2C-SS18 gene fusion and unique histologic and immunophenotypic features characterize a heretofore undefined low-grade salivary adenocarcinoma for which we propose the term "microsecretory adenocarcinoma." RNA-Seq helped establish this entity as a distinct tumor type, and identified one possibly related case with a different SS18-related fusion. The recognition of microsecretory adenocarcinoma and its separation from other adenocarcinomas NOS will facilitate a more complete understanding of the clinical and pathologic characteristics of this previously unrecognized neoplasm.

Programmed death-ligand 1 expression by digital image analysis advances thyroid cancer diagnosis among encapsulated follicular lesions.

Recognition of noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) that distinguishes them from invasive malignant encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) can prevent overtreatment of NIFTP patients. We and others have previously reported that programmed death-ligand 1 (PD-L1) is a useful biomarker in thyroid tumors; however, all reports to date have relied on manual scoring that is time consuming as well as subject to individual bias. Consequently, we developed a digital image analysis (DIA) protocol for cytoplasmic and membranous stain quantitation (ThyApp) and evaluated three tumor sampling methods [Systemic Uniform Random Sampling, hotspot nucleus, and hotspot nucleus/3,3'-Diaminobenzidine (DAB)]. A patient cohort of 153 cases consisting of 48 NIFTP, 44 EFVPTC, 26 benign nodules and 35 encapsulated follicular lesions/neoplasms with lymphocytic thyroiditis (LT) was studied. ThyApp quantitation of PD-L1 expression revealed a significant difference between invasive EFVPTC and NIFTP; but none between NIFTP and benign nodules. ThyApp integrated with hotspot nucleus tumor sampling method demonstrated to be most clinically relevant, consumed least processing time, and eliminated interobserver variance. In conclusion, the fully automatic DIA algorithm developed using a histomorphological approach objectively quantitated PD-L1 expression in encapsulated thyroid neoplasms and outperformed manual scoring in reproducibility and higher efficiency.

Programmed Death - Ligand 1 Expression Distinguishes Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma from Noninvasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features.

BACKGROUND: The noninvasive Encapsulated follicular variant of papillary thyroid cancer (EFVPTC) has been reclassified as Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) without a significant risk for malignant behavior. However the evaluation remains a challenge for clinicians. We sought to determine whether programmed death-ligand 1 (PD-L1) expression may serve as a biomarker to predict invasiveness of EFVPTC and assist to distinguish these neoplasms from NIFTP. METHODS: Immunohistochemical staining of PD-L1 expression was performed in sections of 174 Formalin-fixed paraffin-embedded (FFPE) tissue blocks from surgery removed thyroid nodules. RESULTS: Cytoplasmic PD-L1 expression was significantly increased in invasive EFVPTC (4.76±1.49) as compared to NIFTP (3.06±2.16, p<0.001). Increased cytoplasmic PD-L1 expression was associated with invasiveness in EFVPTC (p<0.001); PD-L1 positive EFVPTC cases were at 3.16 folds higher risk in developing invasion than the PD-L1 negative cases. No significant difference in cytoplasmic PD-L1 expression was observed between NIFTP and benign nodules. CONCLUSION: PD-L1 expression may serve as a useful biomarker in predicting invasiveness of EFVPTC and distinguishing NIFTP from invasive EFVPTC. To our knowledge this is the first report suggesting the application of a protein biomarker to confirm NIFTP as benign indolent 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.

Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants.

Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody (E1L3N, Cell Signaling Technology) and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with p < 0.001 and p < 0.01 respectively). Comparison of PD-L1+ and PD-L1- patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.

ER maleate is a novel anticancer agent in oral cancer: implications for cancer therapy.

ER maleate [10-(3-Aminopropyl)-3, 4-dimethyl-9(10H)-acridinone maleate] identified in a kinome screen was investigated as a novel anticancer agent for oral squamous cell carcinoma (OSCC). Our aim was to demonstrate its anticancer effects, identify putative molecular targets and determine their clinical relevance and investigate its chemosensitization potential for platinum drugs to aid in OSCC management. Biologic effects of ER maleate were determined using oral cancer cell lines in vitro and oral tumor xenografts in vivo. mRNA profiling, real time PCR and western blot revealed ER maleate modulated the expression of polo-like kinase 1 (PLK1) and spleen tyrosine kinase (Syk). Their clinical significance was determined in oral SCC patients by immunohistochemistry and correlated with prognosis by Kaplan-Meier survival and multivariate Cox regression analyses. ER maleate induced cell apoptosis, inhibited proliferation, colony formation, migration and invasion in oral cancer cells. Imagestream analysis revealed cell cycle arrest in G2/M phase and increased polyploidy, unravelling deregulation of cell division and cell death. Mechanistically, ER maleate decreased expression of PLK1 and Syk, induced cleavage of PARP, caspase9 and caspase3, and increased chemosensitivity to carboplatin; significantly suppressed tumor growth and increased antitumor activity of carboplatin in tumor xenografts. ER maleate treated tumor xenografts showed reduced PLK1 and Syk expression. Clinical investigations revealed overexpression of PLK1 and Syk in oral SCC patients that correlated with disease prognosis. Our in vitro and in vivo findings provide a strong rationale for pre-clinical efficacy of ER maleate as a novel anticancer agent and chemosensitizer of platinum drugs for OSCC.

Institution-specific risk of papillary thyroid carcinoma in atypia/follicular lesion of undetermined significance.

BACKGROUND: The Bethesda System for Reporting Thyroid Cytopathology (BSRTC) is used in surgical decision-making according to malignancy risk in each category. Malignancy risk in atypia/follicular lesion of undetermined significance (AUS/FLUS) is estimated in BSRTC to be 5% to 15%, but institutional data have varied widely. METHODS: We conducted a post-BSRTC 4-year retrospective analysis of index thyroid nodule cytology and histopathology in an academic head and neck endocrine surgery setting. RESULTS: Of 2939 thyroid cytology reports from 1944 patients, the most advanced BSRTC category was AUS/FLUS in 233 patients (12.0%) of which 187 went to thyroidectomy. In AUS/FLUS, the upper and lower boundary estimates of the malignancy rate were 46% and 37%, accordingly. The malignancy rate did not vary significantly by cytopathologist or cytopathologic features. CONCLUSION: Malignancy rates in AUS/FLUS vary by institution from 6% to 46%. Given the subjective nature of thyroid cytopathology and interpretation of the BSRTC categories, guidelines should encourage the use of institution-specific data on malignancy risk in treatment decisions. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1210-E1215, 2016.