Faculty Bibliography

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Bodies have continuous reticular networks, comprising collagens, elastin, glycosaminoglycans, and other extracellular matrix components, through all tissues and organs. Fibrous coverings of nerves and blood vessels create structural continuity beyond organ boundaries. We recently validated fluid flow through human fibrous tissues, though whether these interstitial spaces are continuous through the body or discontinuous, confined within individual organs, remains unclear. Here we show evidence for continuity of interstitial spaces using two approaches. Non-biological particles (tattoo pigment, colloidal silver) were tracked within colon and skin interstitial spaces and into adjacent fascia. Hyaluronic acid, a macromolecular component of interstitial spaces, was also visualized. Both techniques demonstrate interstitial continuity within and between organs including within perineurium and vascular adventitia traversing organs and the spaces between them. We suggest that there is a body-wide network of fluid-filled interstitial spaces that has significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.

Background: Thyroseq next-generation sequencing assay and Afirma gene expression classifier (GEC) are used to risk-stratify thyroid nodules with indeterminate cytology: Bethesda III (atypia of undetermined significance, AUS/FLUS) and IV (suspicious for follicular neoplasm, SFN). In this study, we performed a paired comparison of both tests on the same group of indeterminate thyroid nodules with surgical followup.
Design(s): Of 645 AUS/FLUS/SFN cases with both molecular testing and surgical resection in 2014-2017, 40 cases had both Thyroseq (v2) and Afirma GEC performed on the same specimen. Cross-tabulations and ROC curves were created. McNemar tests were done to compare the performance of Thyroseq versus Afirma. The diagnostic performance of combined results were also examined: the combined result was called positive only if both Thyroseq and Afirma were positive/suspicious. Non-invasive follicular thyroid with papillary like nuclear features (NIFTP) on surgical resections was defined as ?positive.? Results: 20/40 (50%) cases were ?positive? on surgical pathology: 8 papillary thyroid carcinoma (PTC), 11 NIFTP, and 1 follicular carcinoma. Thyroseq and Afirma both showed high sensitivity and low specificity in diagnosing malignancy in indeterminate thyroid nodules. Next, the results of both tests were combined. The overall accuracy of combined testing was higher than either test alone (Figure 1). Compared to Afirma alone, the combined test had significantly higher specificity (30% vs 70%, p<0.05, Table 1), while the sensitivity declined from 90% to 75% (p=0.25, Table 1). Compared to Thyroseq alone, there was no significant difference in specificity (45% vs 70% p=0.06) or sensitivity (80% vs 75%, p=1.00, Table 1). Positive predictive value (PPV) improved compared to either test alone. Negative predictive value (NPV) improved compared to Thyroseq alone, and declined only slightly compared to Afirma alone.
Conclusion(s): Molecular testing of cytologically indeterminate thyroid nodules helps determine the extent of surgery. Low diagnostic performance metrics may limit the utility of molecular studies in distinguishing benign from malignant thyroid lesions. Our results show that the combined results of Thyroseq and Afirma improved the specificity and overall accuracy of molecular testing, and provided additional value in the surgical management of patients with indeterminate thyroid nodules. To the best of our knowledge, this is the first study that compares the performance of these two molecular tests on the same thyroid nodules

BACKGROUND AND OBJECTIVES/OBJECTIVE:Carcinoma ex pleomorphic adenoma (CXPA) is a rare disease of the major salivary glands that remains poorly characterized. Our objective was to compare the clinical outcomes of patients with CXPA of the major salivary glands to those with de novo adenocarcinomas. METHODS:Review of the NCDB between 2004 and 2016 to compare cases of CXPA and adenocarcinoma of major salivary glands. Demographics, clinical characteristics, and survival were analyzed. RESULTS:We identified 1181 patients with CXPA and 3326 patients with adenocarcinoma of major salivary glands. Adenocarcinomas presented with higher rates of nodal metastasis (54.7% vs. 30.4%, p < .001). Five-year survival of adenocarcinoma (55.8%) was worse than that of CXPA (68.5%, p < .001). When stratified by nodal status, there was no significant difference in 5-year survival between CXPA and adenocarcinoma node-negative (75.3% vs. 71.6%, respectively) and node-positive (40.4% vs. 36.1%, respectively) patients. CONCLUSIONS:CXPAs of the major salivary glands present at an earlier stage with lower rates of regional metastasis compared to adenocarcinomas. After controlling for lymph node metastases, the outcomes are quite similar.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR₂) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR₂-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared to matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR₂ in Na_V1.8-positive neurons (Par₂Na_v1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par₂Na_v1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR₂-dependent hyperexcitability of trigeminal neurons from WT female mice. Par₂ deletion, LI-1 and inhibitors of adenylyl cyclase or protein kinase A prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N-terminus of PAR₂ at Asn³⁰↓Arg³¹, proximal to the canonical trypsin activation site. Lgmn activated PAR₂ by biased mechanisms in HEK293 cells to induce Ca²⁺ mobilization, cAMP formation and protein kinase A/D activation, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, in the acidified OSCC microenvironment Lgmn activates PAR₂ by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENTOral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR₂) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared to matched normal oral tissue. Lgmn evokes pain-like behavior through PAR₂ Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR₂ Inhibitors of adenylyl cyclase and protein kinase A prevented the effects of Lgmn. Lgmn activated PAR₂ to induce calcium mobilization, cAMP formation and activation of protein kinase D and A, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, Lgmn is a biased agonist of PAR₂ that evokes cancer pain.

In this study, we investigate the feasibility of using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), diffusion weighted imaging (DWI), and dynamic positron emission tomography (PET) for detection of metastatic lymph nodes in head and neck squamous cell carcinoma (HNSCC) cases. Twenty HNSCC patients scheduled for lymph node dissection underwent DCE-MRI, dynamic PET, and DWI using a PET-MR scanner within one week prior to their planned surgery. During surgery, resected nodes were labeled to identify their nodal levels and sent for routine clinical pathology evaluation. Quantitative parameters of metastatic and normal nodes were calculated from DCE-MRI (v_e, v_p, PS, F_p, K^trans), DWI (ADC) and PET (K_i, K₁, k₂, k₃) to assess if an individual or a combination of parameters can classify normal and metastatic lymph nodes accurately. There were 38 normal and 11 metastatic nodes covered by all three imaging methods and confirmed by pathology. 34% of all normal nodes had volumes greater than or equal to the smallest metastatic node while 4 normal nodes had SUV > 4.5. Among the MRI parameters, the median v_p, F_p, PS, and K^trans values of the metastatic lymph nodes were significantly lower (p = <0.05) than those of normal nodes. v_e and ADC did not show any statistical significance. For the dynamic PET parameters, the metastatic nodes had significantly higher k₃ (p value = 8.8 × 10^-8) and K_i (p value = 5.3 × 10^-8) than normal nodes. K₁ and k₂ did not show any statistically significant difference. K_i had the best separation with accuracy = 0.96 (sensitivity = 1, specificity = 0.95) using a cutoff of K_i = 5.3 × 10^-3 mL/cm³/min, while k₃ and volume had accuracy of 0.94 (sensitivity = 0.82, specificity = 0.97) and 0.90 (sensitivity = 0.64, specificity = 0.97) respectively. 100% accuracy can be achieved using a multivariate logistic regression model of MRI parameters after thresholding the data with K_i < 5.3 × 10^-3 mL/cm³/min. The results of this preliminary study suggest that quantitative MRI may provide additional value in distinguishing metastatic nodes, particularly among small nodes, when used together with FDG-PET.

Introduction: Due to the diagnostic dilemma with indeterminate thyroid Bethesda categories III and IV (atypia of undetermined significance, AUS and Suspicious for follicular neoplasm, SFN), many laboratories utilize molecular testing to aid in risk stratification of these nodules. In this study, we evaluated the risk of malignancy (ROM) in AUS and SFN thyroid nodules with subsequent negative molecular (ThyroSeq) test results.
Material(s) and Method(s): This study was designed to evaluate the negative molecular thyroid fine needle aspiration (FNA) cases at a tertiary medical center in the metropolitan area. 109 cases of AUS and SFN thyroid FNAs over 3 years with surgical pathology follow up were included in the study.
Result(s): Of 109 AUS and SFN cases, 4 cases showed insufficient material for ThyroSeq testing (3.7%), 76 cases showed a molecular alteration (69.7%), and 29 cases were negative for an alteration on ThyroSeq (26.6%). Among the cases with negative ThyroSeq results, 26 cases were benign on surgical pathology (89.7%) (7/26 were follicular adenomas), and 3/29 cases were malignant on histopathology (papillary thyroid carcinoma) (ROM=10.3%, Table 1). AUS and SFN cases with molecular alterations showed a significantly higher ROM (ROM= 60.5%) compared to cases testing negative for molecular alterations (p<0.01, z = -4.61).
Conclusion(s): Our study found that indeterminate thyroid nodules that tested negative for a molecular alteration displayed an ROM of 10.3%. This ROM is comparable to the lower limit of ROM of FNA alone (without additional molecular testing data) in the AUS and SFN categories (10-30%), but is significantly lower than the ROM of indeterminate thyroid cases with known molecular mutations. Therefore, clinical follow-up is recommended for thyroid FNA indeterminate nodules, even those testing negative for a molecular alteration, due to the maintained, albeit lower, ROM. [Formula presented]
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Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. PNI induced afterdischarge in A high threshold mechanoreceptors (AHTMRs), mechanical sensitization (i.e., decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low threshold mechanoreceptors (LTMRs). PNI resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically-insensitive and electrically-unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Salivary gland secretory carcinoma, also termed mammary analogue secretory carcinoma (MASC), is a recently described salivary gland neoplasm with characteristic histomorphologic findings similar to those of secretory carcinoma of the breast and harboring recurrent ETV6-NTRK3 fusions. Recent findings have expanded the molecular profile of salivary gland secretory carcinoma to include multiple novel ETV6 fusion partners, including RET, MET, and MAML3. Here, we report a case of cystic MASC with cribriform and papillary histology harboring two gene fusions, ETV6-RET and EGFR-SEPT14, identified by targeted RNA sequencing. The presence of the rearrangements was confirmed by FISH, RT-PCR, and Sanger sequencing. This is the first EGFR-SEPT14 fusion reported in secretory carcinoma as a single event or in association with an ETV6 rearrangement. This finding adds to the expanding molecular profile of this tumor entity, and may translate into novel treatment strategies.