Salivary Research Articles

A non-invasive model for diagnosis of primary Sjogren’s disease based on salivary biomarkers, serum autoantibodies, and Schirmer’s test

BackgroundMinor salivary gland (MSG) biopsy is a critical but invasive method for the classification of primary Sjögren’s disease (pSjD). Here we aimed to identify salivary proteins as potential biomarkers and to establish a non-invasive prediction model for pSjD.MethodsLiquid chromatography-tandem mass spectrometry was conducted on whole saliva samples from patients with pSjD and non-Sjögren control subjects (non-pSjD). Proteins involved in immune processes were upregulated in the pSjD group, such as complement C3 (C3), complement factor B (CFB), clusterin (CLU), calreticulin (CALR), and neutrophil elastase (NE), which were further confirmed by ELISA. Multivariate logistic regression analyses were performed to identify markers that differentiated pSjD from non-pSjD; receiver operating characteristic (ROC) curves were constructed. A diagnostic model based on the combination of salivary biomarkers (CFB, CLU, and NE), serum autoantibodies (anti-SSA /Ro60 and anti-SSA/Ro52), and Schirmer’s test was evaluated in 186 patients (derivation cohort) with replication in 72 patients (validation cohort).ResultsIn multivariate analyses, CFB, CLU, and NE were independent predictors of pSS. A model based on the combination of salivary biomarkers (CFB, CLU, and NE), serum autoantibodies (anti-SSA and anti-Ro52), and Schirmer’s test achieved significant discrimination of pSS. In the derivation cohort, the area under curve (AUC) of the ROC was 0.930 (95% CI 0.877–0.965, P < 0.001), with a sensitivity and specificity of 84.85% and 92.45%, respectively. Notably, similar results were obtained in a validation cohort.ConclusionThe 6-biomarker panel could provide a novel non-invasive tool for the classification of pSjD.

Metastatic Adenoid Cystic Carcinoma to the Pancreas: A Case Report and Review of the Literature.

Although more than 90% of pancreatic tumors represent primary tumors, specifically ductal adenocarcinomas, metastatic tumors are not infrequent at this site. Metastasis of salivary gland tumors to the pancreas is exceedingly rare, with only a few cases reported in the English-language medical literature. Here, we report a patient with metastatic adenoid cystic carcinoma to the pancreas, 24 months following the initial diagnosis, and review the available literature on secondary tumors located in the pancreas with an emphasis on tumors of salivary gland origin.

IFN-γ-producing TH1 cells and dysfunctional regulatory T cells contribute to the pathogenesis of Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disorder characterized by progressive salivary and lacrimal gland dysfunction, inflammation, and destruction, as well as extraglandular manifestations. SjD is associated with autoreactive B and T cells, but its pathophysiology remains incompletely understood. Abnormalities in regulatory T (Treg) cells occur in several autoimmune diseases, but their role in SjD is ambiguous. We had previously shown that the function and development of Treg cells depend on store-operated Ca2+ entry (SOCE), which is mediated by ORAI1 Ca2+ channels and stromal interaction protein 1 (STIM1) and STIM2. Here, we show that mice with a Foxp3+ Treg cell-specific deletion of Stim1 and Stim2 develop a phenotype that fulfills all classification criteria of human SjD. Mutant mice have salivary and lacrimal gland inflammation characterized by strong lymphocyte infiltration and transcriptional signatures dominated by T helper 1 (TH1) and interferon (IFN) signaling. CD4+ T cells from mutant mice are sufficient to induce SjD-like disease in an IFN-γ-dependent manner. Inhibition of IFN signaling with the JAK1/2 inhibitor baricitinib alleviated CD4+ T cell-induced SjD in mice. These findings are consistent with the transcriptional profiles of CD4+ T cells from patients with SjD, which indicate enhanced TH1 but reduced memory Treg cell function. Together, our study provides evidence for a critical role of dysfunctional Treg cells and IFN-γ-producing TH1 cells in the pathogenesis of SjD.

Autocrine TGF-β1 drives tissue-specific differentiation and function of resident NK cells.

Group 1 innate lymphoid cells (ILCs) encompass NK cells and ILC1s, which have non-redundant roles in host protection against pathogens and cancer. Despite their circulating nature, NK cells can establish residency in selected tissues during ontogeny, forming a distinct functional subset. The mechanisms that initiate, maintain, and regulate the conversion of NK cells into tissue-resident NK (trNK) cells are currently not well understood. Here, we identify autocrine transforming growth factor-β (TGF-β) as a cell-autonomous driver for NK cell tissue residency across multiple glandular tissues during development. Cell-intrinsic production of TGF-β was continuously required for the maintenance of trNK cells and synergized with Hobit to enhance cytotoxic function. Whereas autocrine TGF-β was redundant in tumors, our study revealed that NK cell-derived TGF-β allowed the expansion of cytotoxic trNK cells during local infection with murine cytomegalovirus (MCMV) and contributed to viral control in the salivary gland. Collectively, our findings reveal tissue-specific regulation of trNK cell differentiation and function by autocrine TGF-β1, which is relevant for antiviral immunity.

STIMulating salivary glands.

STIMulating salivary glands.

Scanning Electron Microscopic Study on the Connective Tissue Cores of the Lingual Epithelium of the Domestic Chicken (Gallus gallus Domesticus).

Anatomically the tongue of Gallus gallus domesticus (Galliformes: Phasianidae) is distinguished into sharp rostral apex, body and root with numerous conical papillae arranged in rows in different sizes in the free portion and the root of the tongue. The epithelium covered the dorsal and ventral surfaces of the free portion and the dorsal surface of the root of the tongue. Numerous orifices of the salivary glands were scattered in the root of the tongue. SEM investigation of the connective tissue cores after maceration of the tongue in 10% NaOH showed the presence of thread-like structures of the connective tissue cores in the rostral apex of the tongue, rod-shaped protrusions in the middle of the free portion and ring-like process posteriorly, while in the conical papillae, they appeared like thin parallel striations. The connective tissue core formed sheaths around the orifices of the salivary glands. While the connective tissue cores of the laryngeal area showed saw-shaped protrusions. Ventrally, the connective tissue core of the lingual nail was arranged in longitudinal parallel rows. SEM investigation revealed that the connective tissue core of the epithelium covered the tongue and closely conformed to the form and size of the filiform papillae.

Parotid and Sublingual Salivary gland as an Indicator of Antioxidant Efficacy in Experimentally Induced Diabetic Rats

The parotid and sublingual glands were considered the most appropriate tissues to study the effects of diabetes mellitus among the major salivary glands. Globally, diabetes mellitus is a major healthcare concern. It affects bodily tissues and organs in a variety of pathological ways, particularly the major salivary glands. Thirty adult, healthy albino rats were divided into three equal groups of ten animals each. Group I was a control group that did not receive any treatment, group II was experimentally induced to have diabetes mellitus using alloxan, a chemical agent that is more or less safe and permanent, and group III was experimentally induced to have diabetes but treated with ascorbic acid (Vit. C), as an antioxidant drug. All rat groups were sacrificed and tissue samples from their parotid and sublingual glands were prepared. The tissue samples studded histologically using Haematoxylin and Eosin (H&amp;E) stain, histopathologically using Periodic acid Schiff's (PAS) reaction to detect carbohydrates, and immunohistochemically using Beta cell lymphoma-2 (BCL2) as an anti-apoptotic marker. Based on the histology, histopathological, and immunohistochemical significant lesions, the obtained results showed common alterations in the parotid and sublingual glands tissues of diabetic rats. However, compared to untreated animals, rats treated with the antioxidant vitamin C showed fewer tissue changes and lesions. According to the study's findings, vitamin C functions as a helpful antioxidant to assist avoid complications from diabetes.

Misleading Imaging Findings: Bilateral Mylohyoid Defect Presenting as a Submandibular Mass Due to Sublingual Gland Protrusion

Background/Objectives: The muscular base of the oral cavity is formed of the mylohyoid muscle, which forms a sling inferior to the tongue. The muscle is often discontinuous, and defects may include salivary tissues, fat, and/or blood vessels. Hypertrophic sublingual glands located in mylohyoid defects can be herniated into bilateral submandibular spaces and present as palpable masses. The etiology of this condition may be congenital or acquired, and although such anatomical variations are common, they often go unrecognized in clinical practice. Sialoceles are cyst-like structures that result from chronic inflammation or ductal injury, indicating underlying problems with drainage efficiency. Methods: In this case series, we present two patients. Results: The first patient is a 44-year-old female who presented with a slowly enlarging right submandibular mass for two years, while the second is a 70-year-old female who presented with nonspecific neck discomfort, lacking palpable masses. In both, initial imaging (ultrasound and CT) was inconclusive. MRI revealed right sublingual gland herniation through a mylohyoid defect (mylohyoid boutonniere) in both cases. Conclusions: This highlights the importance of comprehensive imaging in the diagnosis of submandibular masses and emphasizes the need for considering mylohyoid boutonniere in cases of bilateral submandibular masses. Further research is warranted into the sialoceles associated with salivary gland abnormalities.

Biochemical Properties of the Acid Ectophosphatase Activity of Phytomonas serpens Involved in Cell Proliferation

Phytomonas is the only kinetoplastid that can parasitize plants, causing economically relevant issues. Phytomonas serpens share similarities with pathogenic trypanosomatids, including surface enzymes that are involved in adhesion to the salivary gland of their experimental host, the insect Oncopeltus fasciatus. Ectophosphatases are cell surface enzymes involved in host–parasite interactions that are widely distributed among microorganisms. This work aimed to perform the biochemical characterization of P. serpens ectophosphatase activity, investigating and discussing its possible physiological role. This activity presented an acidic profile, and its kinetic parameters Km and Vmax were calculated as 1.57 ± 0.08 mM p-NPP and 10.11 ± 0.14 nmol p-NP/(h × 108 flagellates), respectively. It was stimulated by cobalt, inhibited by zinc, and insensitive to EDTA, a divalent metal chelator. The inhibitor sodium orthovanadate was able to decrease P. serpens ectophosphatase activity and growth, suggesting its involvement in cell proliferation. Given that P. serpens can uptake inorganic phosphate (Pi) from the extracellular medium, it is likely that its ectophosphatase activity acts together with the transport systems in the Pi acquisition process. The elucidation of the molecular mechanisms involved in this process emerges as a relevant perspective, providing new strategies for controlling Phytomonas infection.

Application of Human Papillomavirus-DNA Test on Salivary Gland Fine Needle Aspiration Cytology Samples Confirms the Absence of the Virus in Primary Neoplasms and Demonstrates for the First Time Its Presence in Salivary Intraglandular Cysts.

The correlation between Human Papillomavirus (HPV) and salivary gland neoplasms is still controversial. Data in the literature are conflicting, reporting the presence of the HPV-DNA in a significant percentage of cases or none. We investigated the presence of HPV in a series of salivary gland neoplasms using fine needle aspiration cytology (FNAC) samples to explore its potential oncogenic role in salivary gland tumor development. The study included 66 salivary gland lesions from 65 patients. For all cases, residual cytological material in the needle hub, once direct smears were obtained, was fixed by rinsing the needle in Cytolyt hemolytic and preservative solution (Hologic Inc. Marlborough, MA 01752 USA). The samples fixed in the Cytolyt hemolytic and preservative solution (Hologic Inc. Marlborough, MA 01752 USA) were centrifuged at 252 g, then the sediment was transferred to the Preservcyt solution and used for the detection of HPV-DNA. We found evidence of the presence of HPV in two salivary intraglandular cysts lined by squamous cell epithelium. Furthermore, we also found HPV in three metastatic oropharyngeal squamous cell carcinomas (SCC) located in the parotid gland. Regarding primary salivary gland tumors, the HPV test produced negative results in all cases. HPV testing produced negative results in all primary salivary gland tumors, failing to confirm the potential involvement of HPV infection in the pathogenesis of salivary gland tumors. Instead, the presence of HPV DNA in samples of salivary intraglandular cysts, never reported before, may be an interesting fact, which however requires further evaluation.

Insular Infraorbital Neurovascular Pedicle Labial Salivary Gland Transplantation for Severe Dry Eye: Anatomy Study and Case Report.

Severe dry eyes often require surgical intervention. Submandibular salivary and minor salivary gland transplantation are options for refractory dry eyes but have limitations. We innovatively designed an insular infraorbital neurovascular pedicle labial salivary gland transplantation (IINPLSGT) and validated its feasibility and safety through anatomical studies. An anatomical study was conducted on 13 red-colored latex arterial-perfused cadavers (24 sides). Two specimens (4 sides) simulated IINPLSGT. We recorded the branching patterns and courses of the infraorbital artery, infraorbital nerve, and angular artery, as well as the distribution of nerves, vessels, and salivary glands in the upper lip. Infraorbital artery and angular artery were anatomically classified. The feasibility of the IINPLSGT was validated. Otolaryngologists and ophthalmologists performed IINPLSGT, transferring the labial mucosal flap to the lower eyelid conjunctival fornix to treat severe dry eye in 1 patient. Infraorbital artery exhibited 5 potential branches, classified into 5 types based on developmental patterns. Angular artery was classified into 3 types based on the courses. Patient symptoms significantly improved postoperatively, with Schirmer I increasing from 0 mm to 6 mm, noninvasive breakup time from 0 seconds to 6 seconds, and SPEED (Standard Patient Evaluation of Eye Dryness questionnaire) score decreasing from 10.5 to 3 in the OD 1 year after surgery. No severe complications were observed. IINPLSGT is a safe and feasible method for treating severe dry eyes. The procedure is simple, with a high postoperative gland survival rate, stable secretion, and minimal complications.

Surgical treatment of dysphagia due to salivary mucocele (sialocele) in a cat

AbstractThis case report describes a 10‐year‐old, male, neutered, indoor–outdoor, domestic shorthair cat presented for hyporexia and dysphagia of 1‐month duration tentatively attributed to a recent diet modification. The cat was ultimately referred for a second opinion. Examination under sedation confirmed a small, soft, fluctuating mass in the ventral intermandibular space. A fine‐needle aspirate of the mass revealed its contents to be of the salivary gland origin. The sublingual–mandibular salivary gland on the left side was surgically resected, and the left sublingual mucocele (ranula) was marsupialised. Histopathology confirmed a sublingual sialocele of the resected gland. The cat recovered completely, and there has been no recurrence with an 18‐month follow‐up. A sublingual sialocoele is uncommonly seen in cats, but it should be on the differential diagnosis list for dysphagia in cats, especially when a sublingual mass is present.

A rare case of limited granulomatosis with polyangiitis presenting as bilateral parotitis

Background: Granulomatosis with polyangiitis (GPA) is a rare autoimmune vasculitis affecting small and medium-sized vessels, commonly involving the respiratory tract and kidneys. Salivary gland involvement, particularly bilateral parotitis, is an uncommon presentation of GPA. Case Report: We report the case of a 38-year-old Asian male who presented with left ear pain and parotid swelling after a water park visit. Initially treated with antibiotics for suspected otitis externa, his symptoms worsened, leading to bilateral parotitis, facial palsy, and otomastoiditis. Imaging revealed parotid abscesses and lab results showed elevated antineutrophil cytoplasmic antibodies (ANCA), specifically cytoplasmic ANCA directed against proteinase 3, confirming the diagnosis of GPA. Cultures and autoimmune workups for other causes, such as Sjogren’s syndrome and immunoglobulin G4-related disease, were negative. Treatment with high-dose corticosteroids and methotrexate resulted in significant clinical improvement. Conclusion: This case highlights the importance of considering GPA in atypical presentations such as parotitis, even in the absence of renal involvement. Early diagnosis and appropriate immunosuppressive therapy are critical to prevent further complications in multisystem involvement.

Immunoarchitectural Pattern and Its Potential Prognostic Value in Mucoepidermoid Carcinoma.

To define tumor immunoarchitectural patterns (IPs) and characterize the immune profile in salivary gland mucoepidermoid carcinoma (MEC) toward assessing MEC prognostic significance and implications for immunotherapy. This study analyzed 41 MEC cases, evaluating the tumor IPs and tumor-infiltrating lymphocyte (TIL) parameters by using whole-slide imaging and AI-assisted assessment. Immunohistochemistry of CD3 and CD8 markers was performed to assess key lymphocyte subpopulations. Immune-rich (I-rich) tumors were characterized by high TIL density and were associated with aggressive tumor behavior, particularly in histological high-grade MEC, with a significant increase in TIL density observed in the inner invasive margin compartment (p < 0.05). I-rich tumor cases were linked to poorer disease-free survival (DFS) (Breslow = 4.686, p = 0.03). Aggressive MEC cases displayed a highly heterogeneous TIL profile, however, no TIL patterns showed a significant impact on DFS (p > 0.05). Despite the high immunogenicity suggested by the abundance of T lymphocytes, the protective role of CD8+ T lymphocytes was not prominent in MEC. I-rich tumors appeared more aggressive and unfavorable MEC cases exhibited high heterogeneity in their TIL profiles. Interrogating the role of TILs in the inner invasive tumor margin may offer a new mechanistic understanding for future immunotherapy discovery.

TRPS1 Immunohistochemistry in Salivary Gland Neoplasms: Analysis on Cytology Cell Blocks and Surgical Follow-Up Correlation.

In this study we aim to analyze the TRPS1 immunostaining of salivary gland tumors (SGT) on cytology cell blocks and compare the staining pattern on subsequent surgical resections. Malignant SGTs, oncocytomas and basal cell adenomas diagnosed on fine needle aspiration were retrieved from 2019 to 2021 database. Cases with surgical follow-up were selected. TRPS1 staining was performed on cytology cell blocks and surgical specimens. Scoring was interpreted by two pathologists independently. Our cohort comprised of 58 cases: 45 malignant and 13 benign. TRPS1 scoring was interpreted for 44 cytology and 51 surgical cases. 14 cytology cases lacked tumor cells on deeper levels. For 7 cases, surgical blocks were not retrievable. TRPS1 positivity for cytology and surgical cases were 52% and 47% respectively. In the malignant cohort, TRPS1 was positive in 21/32 (66%) cytology cases and 21/43 (56%) surgical cases. All cases of basal cell adenocarcinoma, carcinoma ex pleomorphic adenoma and salivary duct carcinoma were TRPS1 positive on cytology. All cases of adenoid cystic carcinoma and acinic cell carcinoma were TRPS1 positive on surgical resections. In the benign cohort, TRPS1 was positive in 2/12 (17%) cytology cases; however, none of the surgically resected benign cases showed reactivity (0/8). Cytology-surgical correlation of TRPS1 staining was done in 37 cases. We found 21 concordant and 16 discordant cases. Discordance was highest in mucoepidermoid carcinoma. TRPS1 is not an entirely specific marker for breast carcinoma. TRPS1 positivity was noted in a substantial number of salivary gland malignant neoplasms. Cases demonstrating discordance in TRPS1 staining pattern on cytology-surgical correlation warrant further exploration.

A morphological post mortem profile in minor salivary glands changes in females.

Women experience several systemic changes over all stages of life, many due to hormonal fluctuations. The sensation of dry mouth is the most common oral clinical symptom in women from climacteric period onwards, suggesting that sex hormones are important in maintaining salivary glands and oral homeostasis. Although the oral cavity is not conventional considered a target tissue for oestrogen, the extent to which salivary glands morphology and function are impacted by ageing and hormonal variations remain unknown. Due to the clinical demand for understanding oral changes related to the ageing process, this study aimed to portray the glandular morphological aspects in female during the different stages of life. To achieve this, 85 post-mortem specimens of labial minor salivary glands were obtained from females; they were distributed into three groups: group I (reproductive phase: up to 44years, n = 18); group II (climateric phase: 45-55years, n = 21) and group III (post-menopausal phase: over 56years, n = 46). The most significant changes observed included degenerative parenchymal aspects including the replacement of parenchymal structures by fibrous tissue, inflammatory infiltrate as well as vascular and endothelial changes. Ageing was associated with acinar serous metaplasia (p = 0.011), acinar fibrosis (p = 0.042) and ductal ectasia (p = 0.003). The process of acinar atrophy was observed in all groups, with an increase in severity in group III. These degenerative aspects appear to be age-dependent.

Avian malaria parasites (Haemosporida: Plasmodiidae) in mosquitoes (Diptera: Culicidae) of the Curonian Spit (South-East coast of the Baltic Sea)

In recent years, numerous studies have shown an increasing prevalence of avian haemosporidian parasites in Europe. However, little is known about the vectors of these parasites, particularly specific dipterian species transmitting malaria parasites to birds. This study aims to identify vectors of avian malaria parasites on the South-East Baltic coast. Mosquito females were collected from 2020 to 2021 on the Curonian Spit of the Baltic Sea using traps with birds as bait. All insects were identified to the species level through morphological features and the PCR method. Subsequently, they were dissected to extract salivary glands for studying the presence of avian malaria infecting stages (the sporozoites). The remaining mosquito parts were used later for molecular analysis to detect haemosporidian parasite DNA. A total of 596 mosquitoes belonging to 8 species were collected. The analysis revealed that Culex pipiens is a competent vector of avian Plasmodium relictum (genetic lineages pGRW11 and pSGS1) and a potential vector for Plasmodium vaughani (genetic lineage pSYAT05) on the Curonian Spit of the Baltic Sea.

Nicotinamide mononucleotide suppresses cellular senescence and increases aquaporin 5 expression in the submandibular gland of aged male mice to ameliorate aging-related dry mouth.

Dry mouth results from decreased saliva secretion due to aging or drug side effects. Decreased saliva secretion causes dryness in the oral cavity that makes swallowing difficult and increases the risk of aspiration pneumonia. There are few fundamental treatments for dry mouth. Here we investigated whether treatment of old mice with nicotinamide mononucleotide (NMN) improved factors associated with dry mouth. Young (16-week-old) and old (113-week-old) male mice were treated subcutaneously with saline or NMN (300mg/kg) once every two days for four weeks and saliva secretion was measured. The amount of nicotinamide adenine dinucleotide (NAD+) in salivary gland tissues was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene expression in the intestinal tract and salivary glands was measured by real-time PCR. The population of cells with acetylation in the submandibular gland was quantified by immunohistological staining. SA-β-gal activity in the submandibular gland was measured to assess cell senescence. Statistical analysis was performed by one-way analysis of variance with Tukey post hoc analysis. The submandibular glands from old mice treated with NMN exhibited increased saliva secretion and NAD+ levels, which both decrease with aging. In addition, the submandibular glands from NMN-treated old mice had decreased acetylation, numbers of senescent cells, and levels of senescence-associated secretory phenotype (SASP) factors, which all increase with aging, as well as increased aquaporin5 (AQP5) mRNA expression. NMN administration may improve dry mouth by regulating cellular senescence in the submandibular gland and increasing expression of AQP5, a water channel involved in saliva secretion, to inhibit age-related decreases in saliva secretion. It is necessary to elucidate further mechanism and confirm its effectiveness in humans.

Esophageal submucosal gland duct adenoma: An unrecognised esophageal counterpart of minor salivary gland tumours with frequent BRAF V600E mutations.

Esophageal submucosal gland duct adenoma is an extremely rare benign tumour, with only a few reported cases. We conducted the largest single-centre clinical study of esophageal submucosal gland duct adenoma, examining its molecular mechanisms and clinicopathological features. Between 2018 and 2023, seven cases of esophageal submucosal gland duct adenoma were identified at a tertiary medical centre; two were female and five were male, aged between 51 and 75 years (mean = 63.8 years). Comprehensive evaluations of clinicopathological, immunohistochemical and molecular characteristics were conducted. Histologically, tumours showed papillotubular and cystic patterns lined with double layers of cells arranged in ducts, papillary folds and microcysts. The inner luminal tall columnar cells had eosinophilic cytoplasm and did not show mucin production and the basal cells showed myoepithelial differentiation. Immunohistochemically, inner luminal layer cells were positive for CK7, CK19 and CK5/6 and outer basal layer cells were positive for SMA and P40. Both layers were negative for CK20, CDX2, MUC5AC, MUC6, MUC2, GCDFP15 and Alcian blue-periodic acid-Schiff (AB-PAS). Genomic analyses revealed the presence of BRAF V600E mutations in five of seven tumours (71.43%). This study delineates a distinct subtype of benign adenoma arising from the esophageal submucosal gland duct, characterised by multiple lobulated cystic proliferation of benign epithelial layers within the submucosa. BRAF V600E mutations were present, similar to in sialadenoma papilliferum. We determined the genetic mutation present in esophageal submucosal gland duct adenoma, providing further evidence that it is an esophageal counterpart of minor salivary gland tumours.

Targeted RNA sequencing in diagnostically challenging head and neck carcinomas identifies novel MON2::STAT6, NFATC2::NUTM2B, POC5::RAF1, and NSD3::NCOA2 gene fusions.

Although molecular tests developed for a growing list of oncogenic alterations have significantly aided in the classification of head and neck carcinomas, tumours in which prototypical histologic and immunophenotypic features are lacking or only partially developed continue to pose diagnostic challenges. Searching for known diagnostic and therapeutic targets by clinical next-generation sequencing (NGS) assays can often lead to new discoveries. We present our institutional experience in applying targeted RNA NGS in 36 head and neck carcinomas that were morphologically difficult to classify between 2016 and 2023. The patients ranged in age from 5 to 83 years (median, 64), with the majority of tumors occurring in the major salivary glands and the sinonasal tract. Overall, seven (19%) cases showed unusual gene rearrangements, including five novel alterations: MON2::STAT6 in a hard palate adenocarcinoma with mucinous features, POC5::RAF1 in apocrine intraductal carcinoma of the lacrimal gland, EWSR1::CDADC1 fusion in a basaloid carcinoma of the submandibular gland, NFATC2::NUTM2B in myoepithelial carcinoma, and NSD3::NCOA2 fusion in a peculiar high-grade carcinoma with a peritheliomatous growth pattern, and focal myogenic differentiation. Potential therapeutic actionability was identified in three cases (RAF1 and FGFR2 fusions). These findings broaden the current spectrum of gene rearrangements in head and neck carcinomas and support the utility of clinical NGS in identifying unusual, actionable alterations in diagnostically challenging cases.