Recurrent Wnt Pathway and ARID1A Alterations in Sinonasal Olfactory Carcinoma.

Sinonasal tumors with neuroepithelial differentiation, defined by neuroectodermal elements reminiscent of olfactory neuroblastoma (ONB) and epithelial features such as keratin expression or gland formation, are a diagnostically challenging group that has never been formally included in sinonasal tumor classifications. Recently, we documented that most of these neuroepithelial neoplasms have distinctive histologic and immunohistochemical findings and proposed the term "olfactory carcinoma" to describe these tumors. However, the molecular characteristics of olfactory carcinoma have not yet been evaluated. In this study, we performed targeted molecular profiling of 23 sinonasal olfactory carcinomas to further clarify their pathogenesis and classification. All tumors included in this study were composed of high-grade neuroectodermal cells that were positive for pankeratin and at least 1 specific neuroendocrine marker. A significant subset of cases also displayed rosettes and neurofibrillary matrix, intermixed glands with variable cilia, peripheral p63/p40 expression, and S100 protein-positive sustentacular cells. Recurrent oncogenic molecular alterations were identified in 20 tumors, including Wnt pathway alterations affecting CTNNB1 (n = 8) and PPP2R1A (n = 2), ARID1A inactivation (n = 5), RUNX1 mutations (n = 3), and IDH2 hotspot mutations (n = 2). Overall, these findings do demonstrate the presence of recurrent molecular alterations in olfactory carcinoma, although this group of tumors does not appear to be defined by any single mutation. Minimal overlap with alterations previously reported in ONB also adds to histologic and immunohistochemical separation between ONB and olfactory carcinoma. Conversely, these molecular findings enhance the overlap between olfactory carcinoma and sinonasal neuroendocrine carcinomas. A small subset of neuroepithelial tumors might better fit into the superseding molecular category of IDH2-mutant sinonasal carcinoma. At this point, sinonasal neuroendocrine and neuroepithelial tumors may best be regarded as a histologic and molecular spectrum that includes core groups of ONB, olfactory carcinoma, neuroendocrine carcinoma, and IDH2-mutant sinonasal carcinoma.

Mesenchymal Tumors of the Head and Neck.

The majority of neoplasms of the head and neck are of epithelial origin primarily including mucosal squamous cell neoplasms (papillomas; squamous cell carcinoma) as well as salivary gland neoplasms. However, the full spectrum of mesenchymal neoplasms (benign and malignant) typically arising in soft tissue sites may also develop in superficial layers of the upper aerodigestive tract. The diversity of mesenchymal neoplasms arising in the head and neck is beyond the scope of this article, and our focus will be on some of the more common and/or diagnostic problematic mesenchymal tumors occurring in the sinonasal tract, oral cavity/odontogenic, pharynx, larynx, and neck.

Pleomorphic adenoma: the great mimicker of malignancy.

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm, and its diagnosis is straightforward in the majority of cases. However, not infrequently, PA shows unusual and uncommon histological features that can be confused with those of malignancy. The difficulties in diagnosing PA arise from its ability to mimic invasion, show atypical or metaplastic cytomorphology, and show morphological features that overlap with those of established salivary gland carcinomas. In addition, recognising early malignant transformation to carcinoma ex-pleomorphic adenoma continues to be a frequent challenge. This review describes the diagnostic pitfalls of PA, and offers a systematic approach to avoid them by combining classic histopathology with novel immunohistochemical and molecular tests.

Thyroid tumors with follicular architecture.

Thyroid tumors with follicular architecture encompass a considerable array of distinct entities. These lesions share significant morphologic overlap, but portend different prognostic and therapeutic implications. Due to their similar growth patterns, distinction between these tumors can be difficult; remarkable interobserver variability exists, even between expert endocrine pathologists. Given the diagnostic challenges associated with these lesions, establishment of the correct diagnosis requires adequate gross examination protocol, careful attention to morphologic features and pathologic context, as well as-increasingly-adjunct molecular findings. In this review, we summarize the salient features of various follicular thyroid tumors, with special emphasis on the recently defined category of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), as well as the molecular pathology of these lesions.

Virus-associated carcinomas of the head & neck: Update from the 2017 WHO classification.

Virus-associated carcinomas of the head and neck represent an unusual confluence of infections spread by viral transmission and cellular dysregulation resulting in carcinogenesis. While much remains to be elucidated about the exact progression from infection to cancer, a basic framework of viral biology can complement the pathologist's understanding of morphology. This context informs the pathologist's everyday practice, including selecting ancillary studies, communicating prognostically relevant findings, and participating in treatment planning. By comparing and contrasting the salient features of human papillomavirus-associated oropharyngeal carcinoma and Epstein-Barr virus-associated nasopharyngeal carcinoma, this review summarizes recent evidence to guide current practice.

Squamous cell carcinoma of the upper aerodigestive tract: dysplasia and select variants.

Upper aerodigestive tract (UADT) mucosal premalignant lesions include non-keratinizing and keratinizing intraepithelial dysplasia. The keratinizing type of intraepithelial dysplasia represents the majority of UADT dysplasias. Historically, grading of UADT dysplasias has followed a three tier system to include mild, moderate and severe dysplasia. Recent recommendations have introduced a two tier grading scheme to including low-grade (ie, mild dysplasia) and high-grade (moderate and severe dysplasia/carcinoma in situ) providing for better consensus among pathologists in the interpretation of such dysplastic lesions. Squamous cell carcinoma is the most common malignant neoplasm of the UADT. Several variants of squamous cell carcinoma are recognized among which the more common types include papillary squamous cell carcinoma, verrucous carcinoma, spindle cell squamous cell carcinoma (sarcomatoid carcinoma) and basaloid squamous cell carcinoma. Each of these variants of squamous cell carcinoma poses diagnostic challenges and each correlates to specific therapy and prognosis. This review details the proposed update in the grading of UADT dysplasia to a two-tiered system as well as providing the key diagnostic features for select variants of squamous cell carcinoma.

Lymphoepithelial-like carcinomas of the head and neck.

Lymphoepithelial-like carcinomas (LELC) of the head and neck represent malignant neoplasms that are histologically similar to nasopharyngeal carcinoma (NPC), nonkeratinizing undifferentiated type but arise in locations other than the nasopharynx. The most common location for LELC in the head and neck is the salivary glands, in particular the parotid gland. However, LELC may arise in other sites including the oropharynx (tonsils, base of tongue), sinonasal tract, larynx, and middle ear/temporal bone. Unlike the nonkeratinizing undifferentiated type of NPC which is etiologically linked to Epstein-Barr virus (EBV), LELCs are not uniformly associated with EBV. The differential diagnosis for LELC varies per site and depending on the site of occurrence may include lymphoepithelial sialadenitis, sinonasal undifferentiated carcinoma (SNUC), and large cell neuroendocrine carcinoma. Treatment general includes combined (multimodality) therapy including surgical resection, neck dissection, radiation therapy and/or chemotherapy. The prognosis may vary per site overall favorable owing to a good response to therapy.

A unique case of a cutaneous lesion resembling mammary analog secretory carcinoma: a case report and review of the literature.

Mammary analog secretory carcinoma (MASC) is a rare type of salivary gland tumor named for its morphological and genetic similarity to secretory carcinoma of the breast. These tumors are most often found in the parotid gland but have been described in several other mucosal locations of the head and neck. In this case report, a cutaneous lesion most closely resembling MASC was found in a neck mass of a 64-year-old male patient without evidence of a primary salivary gland or oral tumor. The lesion was excised, and the patient remains disease free to date. This case depicts a rare tumor in the skin most closely mimicking MASC and brings additional awareness to dermatopathologists of this tumor.

Evaluation of a new grading system for laryngeal squamous intraepithelial lesions--a proposed unified classification.

AIMS: To verify the applicability, reproducibility and predictive value of a proposed unified classification (amended Ljubljana classification) for laryngeal squamous intraepithelial lesions (SILs). METHODS AND RESULTS: Six internationally recognized experts and three pathologists from Ljubljana contributed to this study by evaluating a set of laryngeal SILs using the new system: low-grade SIL, high-grade SIL, and carcinoma in situ (CIS). The overall agreement among reviewers was good. Overall unweighted and weighted κ-values and 95% confidence intervals were 0.75 (0.65-0.84) and 0.80 (0.71-0.87), respectively. The results were stratified between the international reviewers and the Ljubljana pathologists. The former had good overall agreement, and the latter had very good agreement. Kaplan-Meier survival curves showed a significant difference (P < 0.0001) between patients with low-grade and high-grade SILs; 19 of 1204 patients with low-grade SILs and 30 of 240 patients with high-grade SILs progressed to malignancy in 2-15 years and in 2-26 years, respectively. CONCLUSIONS: The proposed modification to the Ljubljana classification provides clear morphological criteria for defining the prognostic groups. The criteria facilitate better interobserver agreement than previous systems, and the retrospective follow-up study demonstrates a highly significant difference in the risk of malignant progression between low-grade and high-grade SILs.

Pitfalls in the biopsy diagnosis of intraoral minor salivary gland neoplasms: diagnostic considerations and recommended approach.

Among the more common types of intraoral minor salivary gland neoplasms are pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma. These minor salivary gland neoplasms share similar morphologic features and to a large extent immunohistochemical findings. Differentiation between these benign and malignant neoplasms is often predicated on the presence or absence of invasion. As such, in the presence of limited tissue sampling that typifies the initial testing modalities, including fine needle aspiration biopsy and/or incisional biopsy, it often is not possible to differentiate a benign from malignant minor salivary gland neoplasm. The diagnostic difficulties arise from the absence in needle or incisional biopsy of the tumor's periphery to determine whether infiltrative growth is or is not present. In this manuscript we discuss limitations and considerations associated with evaluation of incisional biopsies of intraoral minor salivary gland tumors. We offer a diagnostic approach to evaluating these biopsies, and suggest diagnostic terminology for biopsy specimens in which distinction between benignancy and malignancy is not feasible. The pathologist's approach to this distinction is critical, as treatment of benign neoplasms is generally conservative, whereas malignant lesions may warrant more aggressive management.

RAS-Mutant Follicular Thyroid Tumors: A Continuous Challenge for Pathologists.

The classification of thyroid nodules, particularly those with a follicular growth pattern, has significantly evolved. These tumors, enriched with RAS or RAS-like mutations, remain challenging for pathologists due to variables such as nuclear atypia, invasion, mitotic activity, and tumor necrosis. This review addresses the histological correlates of benign, low-risk, and malignant RAS-mutant thyroid tumors, as well as some difficult-to-classify follicular nodules with worrisome features. One prototypical RAS-mutant nodule is non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). The assessment of nuclear characteristics in encapsulated/well-demarcated non-invasive RAS-mutant follicular-patterned tumors helps distinguish between follicular thyroid adenoma (FTA) and NIFTP. Despite this straightforward concept, questions about the degree of nuclear atypia necessary for the diagnosis of NIFTP are common in clinical practice. The nomenclature of follicular nodules lacking clear invasive features with increased mitotic activity, tumor necrosis, and/or high-risk mutations (e.g., TERT promoter or TP53) remains debated. Invasion, particularly angioinvasion, is the current hallmark of malignancy in RAS-mutant follicular-patterned neoplasms, with follicular thyroid carcinoma (FTC) as the model. Assessing the tumor interface is critical, though full capsule evaluation can be challenging. Multiple levels and NRASQ61R-specific immunohistochemistry can aid in identifying invasion. Controversies around vascular invasion persist, with ancillary stains like CD31, ERG, and CD61 aiding in its evaluation. Moreover, the review highlights that invasive encapsulated follicular variant papillary thyroid carcinoma (IEFVPTC) is closely associated with FTC, suggesting the need for better nomenclature. The concept of "high-grade" differentiated carcinomas, applicable to FTC or IEFVPTC with necrosis and/or high mitotic activity, is also discussed.

Utility of UV Signature Mutations in the Diagnostic Assessment of Metastatic Head and Neck Carcinomas of Unknown Primary.

BACKGROUND: Metastatic carcinoma of unknown primary origin to the head and neck lymph nodes (HNCUP) engenders unique diagnostic considerations. In many cases, the detection of a high-risk human papillomavirus (HR-HPV) unearths an occult oropharyngeal squamous cell carcinoma (SCC). In metastatic HR-HPV-independent carcinomas, other primary sites should be considered, including cutaneous malignancies that can mimic HR-HPV-associated SCC. In this context, ultraviolet (UV) signature mutations, defined as ≥ 60% C→T substitutions with ≥ 5% CC→TT substitutions at dipyrimidine sites, identified in tumors arising on sun exposed areas, are an attractive and underused tool in the setting of metastatic HNCUP. METHODS: A retrospective review of institutional records focused on cases of HR-HPV negative HNCUP was conducted. All cases were subjected to next generation sequencing analysis to assess UV signature mutations. RESULTS: We identified 14 HR-HPV negative metastatic HNCUP to either the cervical or parotid gland lymph nodes, of which, 11 (11/14, 79%) had UV signature mutations, including 4 (4/10, 40%) p16 positive cases. All UV signature mutation positive cases had at least one significant TP53 mutation and greater than 20 unique gene mutations. CONCLUSION: The management of metastatic cutaneous carcinomas significantly differs from other HNCUP especially metastatic HR-HPV-associated SCC; therefore, the observation of a high percentage of C→T with CC →TT substitutions should be routinely incorporated in next generation sequencing reports of HNCUP. UV mutational signatures testing is a robust diagnostic tool that can be utilized in daily clinical practice.

Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: Overview of the 2022 WHO Classification of Head and Neck Neuroendocrine Neoplasms.

This review article provides a brief overview of the new WHO classification by adopting a question-answer model to highlight the spectrum of head and neck neuroendocrine neoplasms which includes epithelial neuroendocrine neoplasms (neuroendocrine tumors and neuroendocrine carcinomas) arising from upper aerodigestive tract and salivary glands, and special neuroendocrine neoplasms including middle ear neuroendocrine tumors (MeNET), ectopic or invasive pituitary neuroendocrine tumors (PitNET; formerly known as pituitary adenoma) and Merkel cell carcinoma as well as non-epithelial neuroendocrine neoplasms (paragangliomas). The new WHO classification follows the IARC/WHO nomenclature framework and restricts the diagnostic term of neuroendocrine carcinoma to poorly differentiated epithelial neuroendocrine neoplasms. In this classification, well-differentiated epithelial neuroendocrine neoplasms are termed as neuroendocrine tumors (NET), and are graded as G1 NET (no necrosis and < 2 mitoses per 2 mm2; Ki67 < 20%), G2 NET (necrosis or 2-10 mitoses per 2 mm2, and Ki67 < 20%) and G3 NET (> 10 mitoses per 2 mm2 or Ki67 > 20%, and absence of poorly differentiated cytomorphology). Neuroendocrine carcinomas (> 10 mitoses per 2 mm2, Ki67 > 20%, and often associated with a Ki67 > 55%) are further subtyped based on cytomorphological characteristics as small cell and large cell neuroendocrine carcinomas. Unlike neuroendocrine carcinomas, head and neck NETs typically show no aberrant p53 expression or loss of RB reactivity. Ectopic or invasive PitNETs are subtyped using pituitary transcription factors (PIT1, TPIT, SF1, GATA3, ER-alpha), hormones and keratins (e.g., CAM5.2). The new classification emphasizes a strict correlation of morphology and immunohistochemical findings in the accurate diagnosis of neuroendocrine neoplasms. A particular emphasis on the role of biomarkers in the confirmation of the neuroendocrine nature of a neoplasm and in the distinction of various neuroendocrine neoplasms is provided by reviewing ancillary tools that are available to pathologists in the diagnostic workup of head and neck neuroendocrine neoplasms. Furthermore, the role of molecular immunohistochemistry in the diagnostic workup of head and neck paragangliomas is discussed. The unmet needs in the field of head and neck neuroendocrine neoplasms are also discussed in this article. The new WHO classification is an important step forward to ensure accurate diagnosis that will also form the basis of ongoing research in this field.

Sclerosing Polycystic Adenoma: Conclusive Clinical and Molecular Evidence of Its Neoplastic Nature.

Sclerosing polycystic adenosis, initially considered a non-neoplastic salivary gland lesion and classified as such in the 2017 WHO Classification of Head and Neck Tumors, has been the subject of controversy regarding its possible neoplastic nature. The reporting of recurrent PI3K pathway alteration represents evidence to support these lesions as being neoplastic and more appropriately referred to as sclerosing polycystic adenoma (SPA). Herein, we provide additional evidence that supports the classification of SPA as a true neoplasm. Eight cases of SPA were identified in our database and consultation files. All cases were subjected to PTEN immunohistochemistry (IHC) and next-generation sequencing (NGS). In addition, one patient underwent genetic counseling and germline testing. The cases included 5 men and 3 women with a mean age of 41 years (range 11-78) and all tumors arose in the parotid gland. One patient had multiple recurrences over a period of 2 years. Morphologically the tumors were circumscribed and characterized by an admixture of acini, ducts and cysts embedded in a fibrotic/sclerotic stroma. The cells lining the ducts and cysts showed variable granular, vacuolated, foamy and apocrine cytoplasmic features, as well as acinar cells contained intracytoplasmic brightly eosinophilic granules. The apocrine intraductal proliferations showed mild to moderate atypia in 6 cases. One case showed overt malignant morphology that ranged from intraductal carcinoma to invasive salivary duct carcinoma. Seven cases tested for PTEN IHC showed loss of nuclear expression in the acinar and ductal cells with retained PTEN expression in the myoepithelial cell and stroma. NGS detected PIK3CA or PIK3R1 genetic alterations in 7 cases, including a novel TFG-PIK3CA fusion. Coexisting PTEN mutations were seen in 4 cases, including in a patient with clinical stigmata of Cowden syndrome and confirmed by germline genetic testing. Our findings herein documented including recurrence of tumor, malignant transformation, high prevalence of PI3K pathway oncogenic alterations and the possible heretofore undescribed association with Cowden syndrome add support to classifying SPA as true neoplasms justifying their designation as adenoma, rather than adenosis.

Sinonasal Tumors With Neuroepithelial Differentiation (Olfactory Carcinoma): Delineation of Their Pathologic and Clinical Features With Insights into Their Relationship to Olfactory Neuroblastoma and Sinonasal Carcinoma.

Olfactory carcinoma is one of many names applied to sinonasal malignancies with histologic similarity to olfactory neuroblastoma (ONB) but cytokeratin expression or gland formation. It is unclear whether these neuroepithelial tumors represent a unified category and if they are separate from ONB and currently-recognized sinonasal carcinomas. This study aims to explore their clinicopathologic characteristics based on a large collective experience. A total of 53 sinonasal tumors with neuroepithelial differentiation were identified affecting 41 men and 12 women, median age 47 years (range: 12 to 82 y). The vast majority arose in the superior nasal cavity and presented at the high Kadish-Morita stage. Frequent histologic findings included (1) lobulated and solid growth, (2) rosettes and/or neurofibrillary stroma, (3) high-grade cytology, (4) complex, often ciliated glands, (5) nonfocal pancytokeratin expression, (6) neuroendocrine pos+itivity, and (7) variable S100-positive sustentacular cells. Twelve patients with available follow-up (48%) developed progressive disease at a median 8 months (range: 0 to 114 mo to progression), and 7 (28%) died of disease. Despite disparate historical terminology, neuroepithelial differentiation is a recurrent and recognizable histologic pattern that is associated with aggressive behavior in sinonasal tumors. While tumors with this phenotype may originate from olfactory mucosa, well-developed epithelial features warrant separation from conventional ONB and neural elements distinguish them from most sinonasal carcinomas. Although their full histogenesis remains uncertain and some heterogeneity may exist, we propose that this pattern is sufficiently distinctive to merit separate recognition as olfactory carcinoma. Use of consistent nomenclature may facilitate greater recognition of tumors with this phenotype and understanding of their pathogenesis and classification.

Phase II Multi-institutional Clinical Trial Result of Concurrent Cetuximab and Nivolumab in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma.

PURPOSE: A phase II multi-institutional clinical trial was conducted to determine overall survival (OS) in patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) treated with a combination of cetuximab and nivolumab. PATIENTS AND METHODS: Patients with R/M HNSCC were treated with cetuximab 500 mg/m2 i.v. on day 14 as a lead-in followed by cetuximab 500 mg/m2 i.v. and nivolumab 240 mg i.v. on day 1 and day 15 of each 28-day cycle. Expression of p16 and programmed cell death-ligand 1 (PD-L1) in archived tumors were determined. Tumor-tissue-modified human papillomavirus (TTMV) DNA was quantified in plasma. RESULTS: Ninety-five patients were enrolled, and 88 patients were evaluable for OS with a median follow-up of 15.9 months. Median OS in the 45 patients who had prior therapy for R/M HNSCC (cohort A) was 11.4 months, with a 1 year OS 50% [90% confidence interval (CI), 0.43-0.57]. Median OS in the 43 patients who had no prior therapy (cohort B) was 20.2 months, with a 1-year OS 66% (90% CI, 0.59-0.71). In the combined cohorts, the p16-negative immunostaining was associated with higher response rate (RR; P = 0.02) but did not impact survival while higher PD-L1 combined positive score was associated with higher RR (P = 0.03) and longer OS (log-rank P = 0.04). In the p16-positive patients, lower median (1,230 copies/mL) TTMV DNA counts were associated with higher RR (P = 0.01) and longer OS compared with higher median (log-rank P = 0.05). CONCLUSIONS: The combination of cetuximab and nivolumab is effective in patients with both previously treated and untreated R/M HNSCC and warrants further evaluation.

The Most Common Mistake in Laryngeal Pathology and How to Avoid it.

Upper aerodigestive tract (UADT) spindle cell squamous carcinoma (SCSC), also known as sarcomatoid carcinoma, is a high-grade subtype of conventional squamous cell carcinoma (SCC) that is histologically characterized by a combination of differentiated SCC in the form of intraepithelial dysplasia and/or invasive differentiated SCC, and the presence of an invasive (submucosal) undifferentiated malignant spindle-shaped and pleomorphic (epithelioid) cell component. Typically, SCSC presents as a superficial polypoid mass not infrequently with surface ulceration precluding identification of an intraepithelial dysplasia. Further, in many cases an invasive differentiated SCC is not identified. Adding to the complexity in such cases, is that immunohistochemical staining in a significant minority of cases is negative for epithelial-related markers but often the cells express mesenchymal-related markers. In such cases, differentiating SCSC from a reactive (benign) spindle cell proliferation or a mucosal-based sarcoma can be problematic, with treatment implications. Herein, we detail the clinical and pathologic features of laryngeal SCSC and discuss the rationale for diagnosing a carcinoma and avoiding a diagnosis of sarcoma. In our experience, such cases represent one of the more common mistakes made in laryngeal pathology. Yet, virtually all such lesions are SCSCs. The treatment and prognosis relies on the accuracy of this distinction.

Middle Ear "Adenoma": a Neuroendocrine Tumor with Predominant L Cell Differentiation.

This morphological and immunohistochemical study demonstrates that tumors currently known as "middle ear adenomas" are truly well-differentiated epithelial neuroendocrine tumors (NETs) composed of cells comparable to normal intestinal L cells, and therefore, these tumors resemble hindgut NETs. These tumors show consistent expression of glucagon, pancreatic polypeptide, PYY, and the transcription factor SATB2, as well as generic neuroendocrine markers and keratins. The same L cell markers are expressed by cells within the normal middle ear epithelium. These markers define a valuable immunohistochemical profile that can be used for differential diagnosis of middle ear neoplasms, particularly in distinguishing epithelial NETs from paragangliomas. The discovery of neuroendocrine cells expressing the same markers in non-neoplastic middle ear mucosa opens new areas of investigation into the physiology of the normal middle ear and the pathophysiology of middle ear disorders.

Molecular Determinants of Thyroid Nodules with Indeterminate Cytology and RAS Mutations.

Background:RAS gene family mutations are the most prevalent in thyroid nodules with indeterminate cytology and are present in a wide spectrum of histological diagnoses. We evaluated differentially expressed genes and signaling pathways across the histological/clinical spectrum of RAS-mutant nodules to determine key molecular determinants associated with a high risk of malignancy. Methods: Sixty-one thyroid nodules with RAS mutations were identified. Based on the histological diagnosis and biological behavior, the nodules were grouped into five categories indicating their degree of malignancy: non-neoplastic appearance, benign neoplasm, indeterminate malignant potential, low-risk cancer, or high-risk cancer. Gene expression profiles of these nodules were determined using the NanoString PanCancer Pathways and IO 360 Panels, and Angiopoietin-2 level was determined by immunohistochemical staining. Results: The analysis of differentially expressed genes using the five categories as supervising parameters unearthed a significant correlation between the degree of malignancy and genes involved in cell cycle and apoptosis (BAX, CCNE2, CDKN2A, CDKN2B, CHEK1, E2F1, GSK3B, NFKB1, and PRKAR2A), PI3K pathway (CCNE2, CSF3, GSKB3, NFKB1, PPP2R2C, and SGK2), and stromal factors (ANGPT2 and DLL4). The expression of Angiopoietin-2 by immunohistochemistry also showed the same trend of increasing expression from non-neoplastic appearance to high-risk cancer (p < 0.0001). Conclusions: The gene expression analysis of RAS-mutant thyroid nodules suggests increasing upregulation of key oncogenic pathways depending on their degree of malignancy and supports the concept of a stepwise progression. The utility of ANGPT2 expression as a potential diagnostic biomarker warrants further evaluation.