Submandibular and sublingual salivary gland involvement in adenoid cystic carcinoma.

Malignant Salivary Gland Tumors

Adenoid cystic carcinoma (AdCC) is a slow-growing salivary gland malignancy that relapses frequently. AdCCs of the submandibular gland exhibit unique differences in prognosis and treatment response to adjuvant radiotherapy compared to other sites, yet the role of tumor anatomic subsite on gene expression and tumor immune microenvironment (TIME) composition remains unclear. We used 87 samples, including 48 samples (27 AdCC and 21 normal salivary gland tissue samples) from 4 publicly available AdCC RNA sequencing datasets, a validation set of 33 minor gland AdCCs, and 39 samples from an in-house cohort (30 AdCC and 9 normal salivary gland samples). RNA sequencing data were used for single sample gene set enrichment analysis and TIME deconvolution. Quantitative PCR and multiplex immunofluorescence were performed on the in-house cohort. Wilcoxon rank-sum, nonparametric equality-of-medians tests and linear regression models were used to evaluate tumor subsite differences. AdCCs of different anatomic subsites including parotid, submandibular, sublingual, and minor salivary glands differed with respect to expression of several key tumorigenic pathways. Among the three major salivary glands, the reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway signature was significantly underexpressed in AdCC of submandibular compared to parotid and sublingual glands while this association was not observed among normal glands. Additionally, the NRF2 pathway, whose expression was associated with favorable overall survival, was overexpressed in AdCCs of parotid gland compared to minor and submandibular glands. The TIME deconvolution identified differences in CD4+ T cell populations between AdCC of major and minor glands and natural killer (NK) cells among AdCC of minor, submandibular, and parotid glands while plasma cells were enriched in normal submandibular glands compared to other normal gland controls. Our data reveal key molecular differences in AdCC of different anatomic subsites. The ROS and NRF2 pathways are underexpressed in submandibular and minor AdCCs compared to parotid gland AdCCs, and NRF2 pathway expression is associated with favorable overall survival. The CD4+ T, NK, and plasma cell populations also vary by tumor subsites, suggesting that the observed submandibular AdCC tumor-intrinsic pathway differences may be responsible for influencing the TIME composition and survival differences.

Tumors of the salivary glands are rare and mainly affect young and middle-aged adults with an equal distribution between the genders 1. The majority of these tumors are of epithelial origin, but while benign tumors are the most common, the carcinomas pose a number of challenges and for some subtypes, namely adenoid cystic carcinoma (ACC), the prognosis may be grave 2. No other organ in the human organism gives rise to such a large spectrum of neoplasia, and the number of different entities has increased dramatically, from 7 carcinoma types in 1972 to 22 in 2017 3, 4. A quite unique feature for this group of tumors is the identification of a network of genes involved in the formation of a variety of fusion oncogenes, each being characteristic of particular tumor types 5. Although believed to be without prognostic significance, these fusion oncogenes have become valuable diagnostic markers and are instrumental in delineating the biology of salivary gland tumors 6. In 1974, Conley and Dingman recapitulate their experience of managing ACC with the following statement: 'Of all tumors in the head and neck region, the adenoid cystic carcinoma is one of the most biologically deceptive and frustrating in management' 7. ACC represents approximately 1% of head and neck malignancies and, in contrast to the rest of the world, it is the most frequent type of salivary gland carcinoma in Denmark 1. It is notorious for its unpredictable and often relentlessly progressive clinical course 2, 8. Although clinically slow growing, ACC is locally invasive and has a propensity for early invasion of peripheral nerves and blood vessels, resulting in a high incidence of local recurrence and distant metastases mainly to the lung, bone, and liver 2, 9. Interestingly, intraneural and not perineural invasion has recently been established to be associated with reduced overall and recurrence-free survival 10. Also, regional recurrence, mainly to the cervical lymph nodes, are highly dependent on the presence of high-grade transformation as well as on the T-site 11, 14, 13, 12. Further complicating the management of ACC patients is the continuing presentation of recurrences many years after primary treatment, causing ACC to constitute a disproportionate disease burden despite its low incidence 2. One reason for this is the shortage of prognostic factors beyond conventional cancer staging and classical histopathological parameters such as close or involved surgical margins and a special histological subset with a predominantly solid growth pattern 2. Metastatic disease is one of the main causes of ACC-related mortality as metastases are often surgically unresectable, are relatively resistant to radiotherapy, and lack effective chemotherapeutic regimens 15-17. Once metastatic disease is diagnosed, median survival is about 2 years but, especially for pulmonary metastases, patients can be asymptomatic for several years 2, 15. If inoperable, normal lung parenchyma is replaced by confluent metastatic masses and this evolution is inevitably fatal. Despite that metastatic disease is one of the main causes of ACC-related mortality, previous research on the biology of ACC has focused on exploring the biology of primary lesions 18-21. Adenoid cystic carcinoma is not a disease that is exclusive to the salivary gland, but is also found in the lacrimal gland and breast, as well as more rare locations including sweat gland, lung, and Bartholin's gland 22-26. Regardless of the site, ACC is characterized by recurrent fusion oncogenes and relatively few but diverse mutations 5, 18, 21, 24, 27-32. Lacrimal gland ACC shares the clinical characteristics of salivary gland ACC, with pronounced infiltrative growth and frequent recurrences in local as well as distant sites 29, 33. In contrast, ACC in the breast is a rare, indolent type of breast cancer, with recurrences and distant spread being vanishingly rare events 34. Also, breast ACC has phenotypic features in common with a subset of breast carcinomas with a very poor prognosis 28. This paradox in the clinical behavior of ACC depending on its origin in the salivary gland or breast has earned ACC the all but flattering title as the Dr. Jekyll and Mr. Hyde of exocrine gland carcinomas 35. But what makes this particular puzzling, besides the identical morphology of ACC in the breast and salivary glands, is that the genetic background, including mutations, copy-number alterations, and gene fusions, is identical 18-21, 32. In this series of studies, we compare the phenotypes, genetics, and microRNA (miRNA) expression profiles of ACC in the salivary gland, lacrimal gland, and breast in order to explore the differences between these seemingly identical tumors with markedly differing prognoses (I). Next, we sought to investigate the genetic and miRNA expressional evolution of salivary gland ACC by comparing paired samples of primary and metastatic lesions (II). Lastly, we performed global miRNA expressional profiling of a large cohort of salivary gland ACC with long-term follow-up to investigate the prognostic value of miRNA (III). In early fetal life, the major salivary glands arise from ectodermal proliferations invading the underlying mesenchyme (Fig. 1) 36. Through a carefully regulated process, these initially solid proliferations develop to form tubuloacinar units consisting of acinar cells, a segmental ductal tree, and myoepithelial cells (Fig. 1). Distinct tumor types arise from each of these compartments, with ACC arising from the intercalated duct segment 37. The salivary glands are divided into (i) major salivary glands, namely the paired parotid-, submandibular-, and sublingual glands, and (ii) minor salivary glands located throughout the mucosal membrane of the upper aerodigestive tract (Fig. 2). The acini form the secretory unit and are composed of inner luminal acinar cells and an outer myoepithelial layer, with the composition of the secretion depending on the site of the acinus producing it. The parotid gland contains almost exclusively serous acinar cells, whereas the palatal minor salivary glands are predominantly of mucous type. Saliva produced by the major and intraoral minor salivary glands facilitates mastication and swallowing and provides lubrication and protection of the mucous membranes and teeth. In addition, saliva contains amylase that initiates digestion of starch, but saliva also plays an essential role in preventing dental caries and infection by direct cleansing of foreign bodies and by an anti-bacterial activity mediated through multiple factors (e.g., IgA and histatins) 38. In contrast, the secretion of the minor salivary glands lining the sinonasal tract is devoid of amylase but mainly functions in lubrication and innate immune defense. Carcinomas of the salivary glands are rare, with an incidence of 1.1/100 000/year in Denmark which has been stable since at least 1990 1. Salivary gland malignancies constitute 0.3% of human cancers and 6% of head and neck cancers in the United States and are predominantly of epithelial origin, with the remaining tumors represented by lymphomas, sarcomas, and metastases mainly to parotid lymph nodes from cutaneous squamous cell carcinomas 39-43. Epithelial salivary gland tumors comprise one of the most diverse groups of tumors in the human organism, with 11 benign and 22 malignant entities listed in the current WHO classification 4. The distribution between benign and malignant tumors varies tremendously according to the gland of origin, with an inverse relationship between the proportion of malignant tumors and the size of the major salivary gland of origin. Accordingly, 17% of parotid gland tumors are malignant, increasing to 38% of submandibular gland tumors and 96% of sublingual gland tumors 44, 45. With the notable exception of the palate, minor salivary gland tumors are malignant in the majority of cases 46, 47. In all surveys on the distribution of different histological types of salivary gland carcinomas, mucoepidermoid carcinoma has been identified as the most frequent 48. However, Denmark is an exception to this, as a nationwide study on Danish salivary gland carcinomas convincingly demonstrated a relatively high incidence of 11 salivary gland carcinomas/1,000,000 Danes/year as well as ACC being the by far the most frequent, constituting more than 25% of all salivary gland carcinomas 1. The reason for this uniquely high proportion of ACC among salivary gland carcinomas in Danes is unknown. Although it is well-known that the Inuit in Greenland (part of the Danish Kingdom) contribute with a high incidence of EBV-related lymphoepithelial carcinoma in salivary glands, these relatively few cases cannot itself explain the higher incidence of salivary gland carcinomas in Danes 1, 49, 50). Epithelial salivary gland tumors are rare in children and adolescents and are, as in adults, predominantly benign 51. In early adulthood, the incidence of benign tumors increases, especially in females, but carcinomas are equally distributed between the genders in Denmark, whereas foreign studies have found a higher incidence of salivary gland carcinomas in males 1, 52, 53. With the exceptions of a markedly increased risk of extranodal marginal zone lymphomas in patients with Sjögren syndrome and salivary gland carcinoma in patients exposed to ionizing radiation, risk factors for and etiology of salivary gland carcinogenesis have not been identified, including in ACC 54, 55. As with all rare diseases, identifying disease-causing factors is complicated due to several factors: First, their rarity makes the accumulation of large patient populations difficult; second, there are no known stages of premalignancy; and third, there has only been identified very few cases of familial accumulation of salivary gland carcinoma that have been identified and no genetic disposition was found in the single case of ACC that underwent genetic testing 56-59. Notable exceptions to the lack of hereditability in salivary gland tumors are the rare hereditary syndromes Muir Torre syndrome (OMIM: 158320), with an increased risk of sebaceous carcinoma, Brooke-Spiegler syndrome (OMIM: 605041), with an increased risk of basal cell adenoma, and Birt–Hogg–Dubé syndrome (OMIM:135150), with an increased risk of oncocytomas 60-62. ACC is considered a sporadic disease. The symptoms caused by salivary gland tumors are very similar across the different histological subtypes and vary according to the anatomical site (Figs 3 and 4). Usually, they are slow growing and fixated to the surrounding tissues. More than 30% of ACCs arise in the parotid gland, and the presenting features for this location are an often painless lump with occasional affection of facial muscle function due to facial nerve invasion 1. The intraoral minor salivary glands constitute the second most frequent site, with more than 25% of ACCs, and this location, along with ACCs in the sinonasal tract and sublingual gland, more frequently cause pain as the presenting symptom due to the propensity of ACC for neural invasion (Fig. 3) 1, 45, 63. Other symptoms of sinonasal ACC include obstructive symptoms, epistaxis, and/or auditory symptoms 64. Ulceration of overlaying mucosa can be seen in oral sites 45, 63. Metastases to bones often cause pain, whereas metachronous pulmonary metastases are often an incidental finding due to chest imaging performed for unrelated reasons. As with other lesions located to the major salivary glands, the preoperative diagnosis is based on the clinical history, imaging, and fine-needle aspiration (FNA) (Fig. 3). FNA has an excellent specificity and good sensitivity in separating benign from malignant lesions in both the major and oral minor salivary glands 65. However, ACC shows occasional cytological overlap with pleomorphic adenoma, basal cell adenocarcinoma, and polymorphous adenocarcinoma 66. For sinonasal lesions, the diagnosis is made by incisional biopsy. The final diagnosis is made by histopathological evaluation of the resected lesion. For diagnostic purposes and for surgical planning, preoperative imaging includes contrast-enhanced computerized tomography (CT) for the identification of bone invasion and/or magnetic resonance imaging (MRI) in assessing soft-tissue extension and the extent of perineural invasion (Fig. 4) 67, 68. The use of fluorodeoxyglucose positron emission tomography (18F-FDG PET) in combination with CT can be employed for the initial staging, and although not all ACCs are PET-positive, the physiological high uptake of the major salivary glands can obscure assessment of the primary lesions (Fig. 4) 69. Of note, caution must be taken in using 18F-FDG PET-CT to exclude distant metastases, especially if the primary tumor does not show enhanced 18F-FDG uptake, which is not uncommon in ACC 69. In a seminal paper published by Robin and Laboulbene in 1853, a tumor of the salivary glands with 'cylindrical' histology and a pronounced tendency to grow along nerves was described for the first time 70. In 1859, Billroth designated this entity as 'cylindroma', a term since modified to adenoid cystic carcinoma by Spies in 1930 71, 72. Regardless of whether it originates in the salivary gland, lacrimal gland, or breast, ACC is a biphasic tumor consisting of relatively uniform ductal and modified myoepithelial cells (Fig. 5). The ductal component is the minor of the two, and the small true lumens formed by these are easily overlooked, as the majority of luminae in ACC are pseudoluminae formed by the dominant myoepithelial component (Fig. 5). The myoepithelial cells lining pseudoluminae produce excess amounts of basophilic, eosinophilic, and occasionally hyalinized extracellular material, often both types varying within a tumor (Fig. 5). The name cylindroma recapitulates the characteristic and most frequent cribriform ('Swiss cheese') growth pattern of ACC, one of the three architectural patterns seen in this malignancy. Tubular and solid patterns are usually seen intermingled with cribriform areas, but often in varying proportions within a single tumor. Especially tubular and cribriform areas usually occur together in the same specimen, and also, in the least frequent of the three, the solid type, minor foci of tubular or cribriform growth are usually present (Fig. 5). Solid ACC, defined as solid growth in >30% of the tumor, is known to have a particularly aggressive clinical course 73. The value of distinguishing tubular and cribriform types is debated, and the WHO defines only the solid type as prognostically relevant, thereby also recognizing the substantial subjectivity in assessing proportions of tubular and cribriform areas in a specimen 74, 75. Invasion of peripheral nerves is a frequent but not specific or universally found hallmark of ACC (Fig. 3). This particular feature enables ACC to invade a significantly larger area than the clinically apparent lesion, with only intraneural invasion being associated with overall and recurrence-free survival (Fig. 3) 10. The immunohistochemical profile of ACC is identical irrespective of anatomical location, with ductal cells being positive for CD117 and the myoepithelial component being positive for p63 and smooth-muscle actin. The proliferation marker ki-67 is positive in a variable proportion of tumor cells and is highest in ACC with solid histology 76. Steroid hormone receptors (estrogen receptor [ER], progesterone receptor [PR], androgen receptor [AR]) and human epidermal growth factor receptor 2 [HER2] are negative, whereas cytokeratin 5/6 (CK5/6) and/or epidermal growth factor receptor [EGFR] have been reported in varying proportions 77-80. Despite its indolent nature, breast ACC belongs to the otherwise aggressive basal-like (estrogen receptor and human epidermal growth factor receptor 2 [HER2] negative, CK5/6 and/or epidermal growth factor receptor [EGFR] positive) and triple-negative (negative for estrogen receptor, progesterone receptor, and HER2) subtypes of breast cancer 35. The phenotypic classification of breast carcinoma (i.e., luminal, basal-like, and HER2 phenotypes) carries significant prognostic and therapeutic implications and has been shown to apply to at least a subset of salivary and lacrimal gland carcinomas 35, 81-83. Interestingly, triple-negative breast carcinoma is generally regarded as a clinically aggressive group of malignancies, in sharp contrast to the case for breast ACC 85, 84. The large number of different types of salivary gland carcinomas are broadly separated into two types: high-risk and low-risk types 86. ACCs, solid as well as tubulocribriform types, are high-grade malignancies, and consequently, the treatment of choice is radical surgical resection and almost always followed by adjuvant radiotherapy (Fig. 4) 8, 87. The surgical approach depends on tumor location and stage at diagnosis, with the objective of tumor-free margins being a compromise between functional outcome and therapeutic benefit 2, 88. The highly infiltrative nature of ACC makes this a complex task to preserve vital structures, speech, and masticatory function, as well as an acceptable cosmetic outcome. There is universal agreement about performing therapeutic neck dissection in the case of either clinically or radiologically detectable involvement of cervical lymph nodes. However, the reported low incidence of occult lymph node metastases has made management of the neck in ACC patients without detectable nodal involvement a matter of controversy 8. While high-grade transformation makes neck dissection mandatory due to a high rate of nodal involvement, recent international collaborations have identified occult lymph node metastases in 17% of clinically node-negative patients, with the highest frequencies found in ACC of the oral cavity (22%) and the lowest in the major salivary glands (12%) 11, 89. Importantly, nodal stage was shown to be an independent prognostic factor for overall and disease-specific survival 89. In head and neck squamous cell carcinoma, the rationale for elective neck dissection in clinically node-negative patients is justified in case of a > 15% risk of occult lymph node metastases 90, 91. Hence, the recommendation of selective neck dissection of levels I–III in clinically node-negative patients, especially if the tumor is located to the oral cavity, now seems justified for therapeutic and staging purposes 2, 92, 89. The effect of radiotherapy in the management of ACC has been a matter of debate. No direct survival benefit is seen in patients receiving adjuvant radiotherapy, but it does improve locoregional control 2, 93. As no data from randomized clinical trials are available, Danish guidelines currently includes adjuvant radiotherapy to all patients with salivary gland ACC 87. Similarly, evidence for the use of chemotherapy, conventional cytotoxic agents, and targeted therapies is heavily lacking. One of the main in these data is the rarity of the the of clinical trials no evidence for treatment, and one must that of the in A approach has shown in other types of salivary gland carcinomas, and recent of to the in patients with ACC to this approach 5, has a in the management of salivary gland ACC to be but this approach has in lacrimal gland ACC asymptomatic patients with disease be the of symptoms and this treatment is to be for patients with symptoms or in the 15. or of the disease may be ACC was first as an its malignant was not due to the course but was regarded as a of the benign pleomorphic due to its to This with the by and in which reported a rate of and distant metastases in of all ACC patients The term for ACC is earned by its relatively good but with recurrences and at least years following primary treatment 93. In a of salivary gland ACCs in Denmark, the and recurrence-free survival was and 2. Similarly, the and overall survival was and 2. factors for salivary gland carcinomas include close or involved surgical and high histological 1. is found between these parameters and shown to have prognostic value in ACC, which also include high close or involved surgical solid histology (i.e., high histological and invasion 2, 10. Importantly, while perineural invasion has been associated with involved surgical it is not associated with prognosis 2, 10. The factors associated with prognosis are not since these features are universally characteristics across human cancer types But while these features are of prognostic across different types of salivary gland carcinoma as well as within there are in based on these features in close or involved margins are found in approximately of patients, invasion is found only in only and a substantial proportion of patients with disease experience and/or distant recurrence 2, Also, the majority of patients metastatic spread are initially with surgical margins are only in of patients, and only have ACC 2, 15. more to a larger proportion of patients are highly The of cancer as a of genetic in a single cell was first by in a that has since been The initially described by in is a term to the of genetic in a including the of genetic from to or from to the early cancer research has identified a of genes that are frequently in cancer, resulting in (i) or (i.e., or (ii) or (i.e., tumor of a growth by mutations, in cancer of these types of genes are and include and which are involved in several cancer In contrast, tumor genes the normal of function, and of these types of genes carries the same effect as of that include the and known to be involved in a variety of different cancer A third, more recently of genes is the of which causes an increased rate of mutations throughout the increasing the risk of mutations in oncogenes and tumor and thereby resulting in cancer formation In the of salivary gland ACC, for mutations has identified only few and diverse mutations in primary tumors as well as the few metastatic lesions 18-21, Regardless of which type of gene is the malignant is the of a variety of by and in as the of cancer being to include cancer is the characteristic namely (i) (ii) growth invasion and of and immune mutations, there are two types of that cause and include and tumor include and and all three of these can in the of fusion genes (Fig. genes their as a of the of two genes by one of two In gene and fusion occur in the of one or both of the involved a fusion gene (Fig. In the gene occur the of both resulting in of the which in increased expression of a normal (Fig. The first fusion described is the arising with formation of the in This fusion gene is formed by the of both the and which is the direct cause of the disease due to the formation of a This the identification of a specific of the fusion which has the prognosis for this patient group types of recurrent fusion genes have been identified across a spectrum of different tumor types, a large proportion of which are whereas are more involved in malignancies fusion genes to be predominantly found in malignancies, but they have since been shown to be frequent in and carcinomas Hence, although gene are not for specific they have in tumor and as for the of targeted therapies 5. In salivary gland carcinomas, the most well-known with direct therapeutic is the of HER2 in salivary duct carcinoma, in which treatment has shown some effect in a subset of patients However, resulting in the formation of recurrent fusion genes have been shown to occur in a larger number of different types of salivary gland carcinomas 5, the prognostic value has been for some of especially the in mucoepidermoid carcinoma, but namely the in secretory carcinoma and in a subset of salivary duct carcinoma, are with good reported In ACC, the most frequent fusion gene of the of the to the of the factor which in most cases is the primary in ACC formation (Figs and to and different in both multiple have been with most the first of and from 11 of 21, very few have been described (Fig. Interestingly, a substantial proportion of ACCs without also the which is due to the of expression an fusion between the and was shown to occur in a substantial proportion of ACCs without fusion The and genes substantial in their functional and the fusion includes the first of similar to the involvement of in the fusion However, while identification of these gene are valuable diagnostic for ACC, only very few of these cases have been reported and the prognostic value is 21, no targeted therapies are for gene fusion found in plays a role in the formation of human and this has to to many in However, the of in biology was more than years and since several gene expression have been identified, including by of and gene by especially by miRNA to the involvement of these genes in cancer is that genes involved in these are frequently in human cancer In the of it is that miRNA genes are frequently located at sites and frequently involved in cancer, a

Salivary function provides host protection, assists in the initiation of food and fluid intake, and enables communication through speech. Without adequate salivary output, oral and pharyngeal health declines along with a person’s quality of life. The complaint of a dry mouth (xerostomia) and the objective finding of salivary dysfunction are common occurrences in older individuals, producing transient and permanent oral and systemic problems. Salivary dysfunction, however, is not a normal consequence of growing older, and is due to systemic diseases, medications, and head and neck radiotherapy. Diagnosis of salivary disorders begins with a careful medical history, head, and neck examination. While complaints of xerostomia may be indicative of a salivary gland disorder, salivary diseases can present without symptoms. Therefore, routine examination of salivary function must be part of any head, neck, and oral examination. Therapies are designed to prevent the development of oral and pharyngeal sequelae of salivary hypofunction. Current xerostomia-based treatments include replacement therapies and gustatory, masticatory, and pharmacological stimulants. Healthcare professionals can play a vital role in identifying patients at risk for developing salivary dysfunction, and should provide appropriate preventative and interventive techniques that will help preserve a person’s health, function, and quality of life.

The salivary glands are divided into major salivary glands, consisting of the paired parotid, submandibular, and sublingual glands, and the minor salivary glands, composed of the 600 to 1000 glands distributed throughout the upper aerodigestive tract. Because of the diversity of tumor types and the relative rarity of salivary gland neoplasm, exact diagnosis and treatment decision have been difficult. Treatment was surgical and the resection was conservative when possible, depending upon the extent of the tumor. Seventy cases of salivary gland tumors which were diagnosed at our hospital from January 1987 to December were reviewed according to age, sex, site, presenting symptoms, staging, histology, and outcome, retrospectively. The following results were obtained; There were 27 males(38% ) and 43 females(62% ). The male to female ratio was 1 : 1.5 in benign tumors, and 1 : 2 in malignant tumors. Of all salivary gland tumors, 35 cases(50%) arose in the parotid gland, 16 cases(23%) in the submandibular gland, and 19 cases(27%) in the minor salivary glands. Among the benign tumors, size of 1 ∼3cm was 31 cases(56% ). There were 55(79%) benign tumors, and 15(21%) malignant tumors. The most frequent benign tumor was pleomorphic adenoma(89% ), and malignant tumor was adenoid cystic carcinoma(400. The incidence of malignancy was 11% in parotid gland, 31% in submandibular gland, and 32% in minor glands. The symptoms varied in duration from several weeks to 26 years. 29 cases(41%), had symptoms for one to five years. The most common presenting symptom was asymptomatic slowly growing mass(54%) followed by pain, rapidly growing mass, rhinorrhea, nasal obstruction, and facial nerve paralysis. The incidence of neck node involvement was 27% (4 cases). According to TNM system of AJC, 3, 5, 2, and 5 cases had stages I through IV. Superficial parotidectomy was performed 21 cases(60%) of parotid tumor. Among the submandibular and minor salivary glands, wide excision was performed 13 cases(81%) and 14 cases(74%). Among the parotid and submandibular glands, postoperative facial nerve paralysis was occurred 9 cases(18%). Recurrence was seen in 4 cases(6%).

Background and Objectives: Mucin has been implicated via various mechanisms in the development and growth of tumour cells. However, mucin expression studies in salivary gland tumours are limited, especially with samples from minor salivary glands. This study aims to investigate and compare mucin expression in benign and malignant salivary gland tumours of minor and major salivary gland origins. Materials and Methods: Special stains were used to stain neutral mucin (Periodic acid Schiff), sialomucin (Alcian Blue) and sulfomucin (Aldehyde Fuschin) within tissues from six normal salivary glands and 73 salivary gland tumours including 31 pleomorphic adenomas, 27 mucoepidermoid carcinomas, and 15 adenoid cystic carcinomas. A semi-quantitative approach was used to evaluate mucin expression within ductal lumens. Sialomucin was the most expressed mucin in all salivary gland tumours, regardless of origin. Results: A significant difference was observed in the mucin expression between benign and malignant salivary gland tumours, as pleomorphic adenoma showed three times significantly higher expression of sialomucin compared to mucoepidermoid carcinoma and adenoid cystic carcinoma (p = 0.028). Pleomorphic adenomas of major glands showed 42 times significantly higher expression of sialomucin compared to those of minor glands (p = 0.000). Conclusions: Sialomucin content in pleomorphic adenomas of major glands was vastly increased compared to that in minor glands. Differential sialomucin expression in benign and malignant salivary gland tumours suggests a role in diagnosing of borderline salivary gland tumours.

Update on Diagnostic Difficulties in Lesions of the Minor Salivary Glands

Adenoid cystic carcinoma (AdCC) is a malignant salivary gland tumor. To date, no cases of AdCC in first-degree relatives have been reported in the literature. We present a 50-year-old female (Case 1) and this patients' father (Case 2), both of whom were diagnosed with AdCC of the minor salivary glands. Histology of Case 1 demonstrated a tubulocribriform AdCC whereas Case 2 primarily was an AdCC of solid type. Both cases harbored the MYB-NFIB gene fusion as demonstrated by FISH and RNA-sequencing. After filtering and selection of putative deleterious variants, whole exome sequencing identified 18 germline variants in common between Case 1 and Case 2. However, none of the variants were associated with AdCC or other head and neck cancers. To our knowledge, we present the first potential case of familial AdCC. The presented genetic data may contribute to further investigations of the underlying genetic mechanisms for AdCC susceptibility.

To investigate the incidence and clinicopathologic features of cervical lymph node metastasis in salivary gland adenoid cystic carcinoma (AdCC). Retrospective analysis was made in 798 AdCC patients who underwent tumor resection during January, 2001 to January, 2019 in Peking University School and Hospital of Stomatology, especially the clincopathologic characteristics of 82 cases with lymph node metastasis. In the study, 82 of the 798 patients were identified with cervical lymph node metastasis, which was confirmed by histopathological examination. The palate, the region of sublingual gland and mouth floor, and the parotid were the frequently involved primary sites for AdCCs. The general incidence rate of lymph node metastasis was approximately 10%. The submandibular gland, the region of sublingual gland and mouth floor, and the mobile tongue were the most frequent sites of lymph node metastasis with the incidence rates of 20.8%, 16.1%, and 15.1%, respectively; while lymph node metastasis was uncommon in the tumors which were the origin from the palate and parotid, with incidence rates of 6.1% and 3.4%, respectively. Most AdCC cases (70.7%) showed the classic "tunnel-style" metastatic pattern of occurrence, and the level I and II regions were the most frequently involved areas. The 5-year and 10-year overall survival rates of the patients with lymph node metastasis were 77.4% and 20.6% respectively, while the 5-year and 10-year overall survival rates of the patients with no lymph node metastasis were 83.5% and 57.6%, respectively. The univariate analysis demonstrated that statistically significant differences in the overall survival for the presence of lymph node metastasis (P<0.001). In the meantime, the 5-year disease-free survival rate of the patients with lymph node metastasis also showed statistically significant differences to that of the AdCC patients with no lymph node metastasis. In addition, the primary site and histological grade were significantly associated with lymph node metastasis, and the high-grade solid growth pattern was identified as a strong predictor for the occurrence of lymph node metastasis. Cervical lymph node metastasis has a high tendency of occurrence in submandibular gland and tongue-mouth floor complex, and the high-grade solid growth pattern could be taken as a strong predictor for the occurrence of lymph node metastasis, which correlates to poor prognosis of AdCC patients. A selective neck dissection should be considered as a management in such patients.

Background: Adenoid cystic carcinoma (ACC) is an uncommon tumor with nonspecific clinicoradiological features thereby masquerading other nonneoplastic and neoplastic entities. Materials and Methods: Cases of ACC were retrospectively reviewed over a period of 4 years. The clinical details of these patients including fine-needle aspiration cytology (FNAC) and imaging findings were retrieved. Diagnosis was confirmed on histomorphology and supplemented with immunohistochemistry (IHC). Results:: Thirty cases of ACC were included in the study. Mean patient age was 55.5 years with a slight female preponderance. Among the 30 ACCs, 10 (33.4%) were located in submandibular gland, 7 (23.4%) in parotid gland, 6 (20%) in sublingual gland, 2 (6.7%) in lung and one each (3.33%) in nasal cavity, breast, cervix, lip, and skin of face. Preoperative imaging was suggestive of malignancy in 29 cases while a single case of parotid gland ACC was misdiagnosed as benign salivary gland neoplasm. FNAC was performed in 29 cases with a diagnostic accuracy of 82.7%. Histopathological examination showed characteristic features of ACC in all cases with perineural invasion seen in 7 cases. On IHC, positivity for cytokeratin was seen in all cases, cluster of differentiation 117 in 24 cases, thyroid transcription factor-1 in two cases and human epidermal growth factor receptor/neu in two cases. All cases were negative for estrogen receptor and progesterone receptor IHC. Mean Ki-67 score was 47.8%. Conclusion: ACCs are notorious tumors showing slow growth kinetics with propensity for perineural invasion, late recurrences, and distant metastasis. It should be kept in mind as a differential diagnosis at unusual sites other than salivary glands.

Adenoid cystic carcinoma (AdCC) is a common malignant tumour of the salivary gland characterised clinically by slow progression but a high rate of recurrence and metastasis. AdCC consists of two types of cells, ductal epithelial cells and myoepithelial cells, with three main growth patterns: tubular, sieve and solid.1Stennman G. Licitra L. Said-AI-Naief N. et al.Adenoid cystic carcinoma.in: El-Naggar A.K. Chan J.K. Grandis J.R. WHO Classification of Head and Neck Tumours. 4th ed. IARC Press, Lyon2017: 164-165Google Scholar The occurrence of high-grade transformation (HGT) in AdCC is a rare phenomenon, with the high-grade component most often showing hypodifferentiated adenocarcinoma or undifferentiated carcinoma, with histological features including loss of the biphasic pattern and typical histopathological features of malignancy, such as marked cellular atypia, increased proliferative activity and necrosis. It is considered a highly aggressive tumour because of its high recurrence rate and risk of metastasis and poor prognosis.2Seethala R.R. Hunt J.L. Baloch Z.W. et al.Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature.Am J Surg Pathol. 2007; 31: 1683-1694Crossref PubMed Scopus (192) Google Scholar Here, we report a rare case of adenoid cystic carcinoma with high-grade transformation (AdCC-HGT). An 80-year-old female presented with a swelling on the right side of her tongue that was self-reported more than 10 years previously and had increased in size over the past year. Enhanced computed tomography (CT) of the head and neck revealed a slightly dense shadow in the tip of the tongue, which showed significant enhancement. A small biopsy was performed in another hospital and the result was reported to be a malignant tumour of minor salivary gland origin with a tendency to pleomorphic adenocarcinoma. The patient underwent partial resection of tongue tumour and cervical lymph node dissection. An intraoperative cauliflower-like mass of about 2.5×1.5 cm in size, hard and poorly defined, without involvement of the floor of the mouth, was seen on the right side of the tongue. The surgical specimen sent for examination was a piece of grey-red irregular tissue measuring 3.3×2.5×2.3 cm, and a greyish-white nodule measuring 2.0×1.0×0.7 cm, with moderate solidity and still-clear borders, which were visible on the cut surface. After haematoxylin-eosin staining, the tumour was seen to consist of two parts under the microscope (Fig. 1A). In one part, the tumour cells consisted of luminal surface cells and myoepithelial cells, arranged in small clusters and stripes, with small foci in the shape of sieves. Basophilic mucus was seen in the pseudocapsular cavity, with abundant extracellular matrix, and glassy interstitial fibres which squeezed the tumour cells into a bundle-like striped arrangement (Fig. 1B). In the other part, the tumour was arranged in a large solid nest-like pattern, with lamellar necrosis seen in the centre of the nest. Compared with the former, the tumour cells were clearly heterogeneous, with large, vacuolated nuclei, obvious nucleoli, visible nuclear schizophrenia, and reduced or absent myoepithelium (Fig. 1C). A zone of gradual transition existed between these two areas. The tumour invaded the nerves, and tumour metastases were visible in the lymph nodes (Fig. 1D). Immunohistochemical staining showed myoepithelial expression of p63, p40, S100 and calponin in a portion of the tumour area (Fig. 2A,B), p53 variable strong positive expression in 40% of the tumour cells (Fig. 2C), and Ki-67 positivity in about 40% (Fig. 2D). In the other part, no myoepithelium was seen (Fig. 2A,B), p53 showed strong positive expression in 80% of tumour cells in a diffuse and consistent manner (Fig. 2C), and Ki-67 was positive in about 85% (Fig. 2D). Fluorescence in situ hybridisation (FISH) assay revealed a break in the MYB gene. The final diagnosis was AdCC-HGT, infiltrating into the transverse muscle, positive for nerve involvement, negative for vascular involvement, negative for cut margins, and cancer metastasis visible in lymph nodes (7/19). This case was treated with radiation therapy three times after surgical resection and followed up for 18 months, with no recurrence or metastasis in survival to date. More than 60 cases of AdCC-HGT have been reported in the literature2Seethala R.R. Hunt J.L. Baloch Z.W. et al.Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature.Am J Surg Pathol. 2007; 31: 1683-1694Crossref PubMed Scopus (192) Google Scholar, 3Cheuk W. Chan J.K. Ngan R.K. Dedifferentiation in adenoid cystic carcinoma of salivary gland: an uncommon complication associated with an accelerated clinical course.Am J Surg Pathol. 1999; 23: 465-472Crossref PubMed Scopus (126) Google Scholar, 4Hellquist H. Skálová A. Barnes L. et al.Cervical lymph node metastasis in high-grade transformation of head and neck adenoid cystic carcinoma: a collective international review.Adv Ther. 2016; 33: 357-368Crossref PubMed Scopus (51) Google Scholar, 5Tando S. Nagao T. Kayano K. et al.High-grade transformation/dedifferentiation of an adenoid cystic carcinoma of the minor salivary gland to myoepithelial carcinoma.Pathol Int. 2018; 68: 133-138Crossref PubMed Scopus (9) Google Scholar, 6Miyazaki M. Aoki M. Koga K. et al.Adenoid cystic carcinoma with high-grade transformation forming spindle cell component of the submandibular gland.Auris Nasus Larynx. 2019; 46: 934-939Abstract Full Text Full Text PDF PubMed Google Scholar, 7Chia N. Petersson F. Adenoid cystic carcinoma with dedifferentiation/expansion of the luminal cell component and preserved biphasic morphology - early high-grade transformation.Ann Diagn Pathol. 2021; 50151650Crossref PubMed Scopus (5) Google Scholar since Cheuk et al. first described the ‘dedifferentiation’ of AdCC in 1999,3Cheuk W. Chan J.K. Ngan R.K. Dedifferentiation in adenoid cystic carcinoma of salivary gland: an uncommon complication associated with an accelerated clinical course.Am J Surg Pathol. 1999; 23: 465-472Crossref PubMed Scopus (126) Google Scholar followed by Seethala et al. who first systematically described the pathological features of high-grade transformation and generalised the diagnostic criteria for high-grade transformation of AdCC.2Seethala R.R. Hunt J.L. Baloch Z.W. et al.Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature.Am J Surg Pathol. 2007; 31: 1683-1694Crossref PubMed Scopus (192) Google Scholar In this case, the fibrous interstitium was sclerotic and most of the solid AdCC areas were extruded in a striated pattern. This case was mistakenly thought to be a ‘polymorphic adenocarcinoma’ (PAC) by another hospital before immunohistochemical staining, due to the limited biopsy of small specimens. PAC tumors are structurally diverse, trabecular and sieve-like are common, and sometimes have a morphology similar to that of AdCC, but the swirling, target-ring-like arrangement of the peripheral borders of the tumors is the characteristic structure.8Fonseca I. Assaad A. Katabi N. et al.Polymorphous adenocarcinoma.in: El-Naggar A.K. Chan J.K. Grandis J.R. WHO Classification of Head and Neck Tumours. 4th ed. IARC Press, Lyon2017: 167-168Google Scholar Moreover, AdCC ducts are composed of bilayer cells and immunohistochemical staining is positive for both p63 and p40, whereas PAC is positive for p63 and often negative for p40,1Stennman G. Licitra L. Said-AI-Naief N. et al.Adenoid cystic carcinoma.in: El-Naggar A.K. Chan J.K. Grandis J.R. WHO Classification of Head and Neck Tumours. 4th ed. IARC Press, Lyon2017: 164-165Google Scholar,8Fonseca I. Assaad A. Katabi N. et al.Polymorphous adenocarcinoma.in: El-Naggar A.K. Chan J.K. Grandis J.R. WHO Classification of Head and Neck Tumours. 4th ed. IARC Press, Lyon2017: 167-168Google Scholar which will help to differentiate the two. It should be noted that the high-grade transformation zone of AdCC-HGT needs to be distinguished from solid type AdCC. In this case, the tumour cells in the high-grade transformation zone had significantly increased heterogeneity and more nuclear divisions, necrosis was seen in the centre of the tumour nest, and immunohistochemistry could confirm the features of adenocarcinoma such as loss of myoepithelium and elevated Ki-67, all of which helped to confirm the high-grade transformation. Similar to a case reported by Tando et al.,5Tando S. Nagao T. Kayano K. et al.High-grade transformation/dedifferentiation of an adenoid cystic carcinoma of the minor salivary gland to myoepithelial carcinoma.Pathol Int. 2018; 68: 133-138Crossref PubMed Scopus (9) Google Scholar our case also had a typical transition zone between the conventional and highly malignant tumour components, suggesting the same origin and a possible manifestation of malignant progression. By immunohistochemical staining, p53 showed wild-type in the conventional AdCC region and missense mutant in the HGT region. Due to the small number of cases, there are not many studies on the mechanisms of molecular biological alterations in AdCC-HGT, and some molecular studies have reported TP53 mutations and overexpression of p53 protein in high-grade components,9Chau Y. Hongyo T. Aozasa K. et al.Dedifferentiation of adenoid cystic carcinoma: report of a case implicating p53 gene mutation.Hum Pathol. 2001; 32: 1403-1407Crossref PubMed Scopus (70) Google Scholar,10Seethala R.R. Cieply K. Barnes E.L. et al.Progressive genetic alterations of adenoid cystic carcinoma with high-grade transformation.Arch Pathol Lab Med. 2011; 135: 123-130Crossref PubMed Google Scholar suggesting that p53 abnormalities may play a major role in the development of AdCC-HGT. A break in the MYB gene was found in this case by FISH, whereas alterations in the MYB gene were rare in previous reports on AdCC-HGT. Chromosomal translocation of MYB, which is considered an early and major oncogenic factor in AdCC, and this event occurs in approximately one-third of AdCC.11Mitani Y. Li J. Rao P.H. et al.Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: incidence, variability, and clinicopathologic significance.Clin Cancer Res. 2010; 16: 4722-4731Crossref PubMed Scopus (237) Google Scholar Although the study of MYB in AdCC by Costa et al. showed unique genetic alterations in both conventional and transformed components,12Costa A.F. Altemani A. García-Inclán C. et al.Analysis of MYB oncogene in transformed adenoid cystic carcinomas reveals distinct pathways of tumor progression.Lab Invest. 2014; 94: 692-702Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar the presence of alterations in the MYB gene in clinical cases still contributes to the diagnosis of AdCC-HGT. Similar to conventional AdCC, nerve invasion is common in the HGT region, but AdCC-HGT usually results in metastasis to the cervical lymph nodes in about half of cases, which is 5–10 times more common than conventional types of AdCC.4Hellquist H. Skálová A. Barnes L. et al.Cervical lymph node metastasis in high-grade transformation of head and neck adenoid cystic carcinoma: a collective international review.Adv Ther. 2016; 33: 357-368Crossref PubMed Scopus (51) Google Scholar In this case, in addition to nerve invasion, metastatic carcinoma was found in nearly half of the cervical lymph nodes, and most of them had a high-grade transformed form. Due to the high proliferative activity and lymph node metastasis rate, three radiation treatments were performed after surgical resection and regional lymph node dissection in this case. After 18 months of follow-up we have not seen tumour recurrence or metastasis, although the median survival time for AdCC-HGT is 36 months as shown through previous case reports.2Seethala R.R. Hunt J.L. Baloch Z.W. et al.Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature.Am J Surg Pathol. 2007; 31: 1683-1694Crossref PubMed Scopus (192) Google Scholar In conclusion, AdCC-HGT is a relatively rare and more malignant phenomenon, which should not be missed by misdiagnosing the transformed area as solid type, and sometimes needs to be differentiated from PAC, where high-grade transformation is more common. It is helpful for the diagnosis of AdCC-HGT if immunohistochemical staining shows myoepithelial deletion and molecular detection shows MYB mutation. The authors state that there are no conflicts of interest or sources of funding to disclose.

Estrogen receptor overexpression in malignant minor salivary gland tumors of the sinonasal tract

The aim of this study was to investigate the epidemiological and clinical characteristics of salivary gland tumors (SGTs) in a Taiwanese population and to provide data for comparison with other studies in various locations and racial populations. We retrospectively analyzed the medical records of 559 patients with tumors of the salivary glands who were surgically treated from 2002 to 2017 at a single institution. There were 430 benign and 129 malignant tumors with a mean age of 53.5 years (range, 1-91 years). The mean age of patients with benign SGTs was 52.6 years, and the mean age of patients with malignant SGTs was 55.8 years. Most findings were similar to those reported in literature, with some variations. The salivary tumors slightly predominated in males. There were no differences in age and incidence of benign or malignant major SGTs between males and females. The frequency was 76.9% for benign tumors (430 patients) and 23.1% for malignant tumors (129 patients). The majority of the tumors occurred in the parotid gland (351 cases), followed by the submandibular gland (152 cases), the minor salivary glands (46 cases), and, lastly, the sublingual gland (10 cases). Minor SGTs occurred most frequently on the palate, with pleomorphic adenoma, the most frequent benign tumor type, and adenoid cystic carcinoma and mucoepidermoid carcinoma, the commonest malignant tumor types. Tumors of the sublingual gland were rare, but 80% were malignant. In our series, SGTs were uncommon neoplasms that typically arose in the parotid gland. Benign tumors were far more common than malignant tumors. Pleomorphic adenoma was the most common benign tumors. Adenoid cystic carcinoma and mucoepidermoid carcinoma constituted the most common malignancies. Almost 60% of the tumors arising from minor salivary glands were malignant; thus, special care must be taken when these glands are affected.

Background Incisional biopsy is indicated for intraoral tumors, but it is a contraindicated surgical procedure for major salivary glands. To avoid complications and facilitate diagnosis, fine needle aspiration biopsy (FNAB) is a type of biopsy widely used for preoperative diagnosis in these glands.Material and Methods The aim of this study was to analyze the agreement between the diagnosis by FNAB (major glands), incisional biopsy (minor glands) and histopathological analysis of the surgical specimen in salivary gland tumors from a database (medical records) of patients treated in a cancer treatment reference center in the Northeast region of Brazil.Results The sample consisted of 110 cases, being 86 of them malignant tumors in major salivary glands (parotid gland=73; submandibular gland=13) and 24 cases in minor salivary glands (palate). The female gender was the most affected (57.3%), especially in patients over 60 years (42.7%). In the TNM classification, 41.8% of the cases were in T2 at the time of diagnosis, with most of the regional lymph nodes in N0 (85.5%) and 87.3% of the cases in M0. FNAB was able to identify malignant neoplasms in 68.6% of the cases (n=59), while incisional biopsy in palatal tumors obtained agreement of 75% of the cases (n=18). The analysis revealed that tumors classified as T3-T4 (p=0.012) showed greater agreement between pre- and post-surgical diagnosis.Conclusions The results obtained in this study indicate that FNAB has similar accuracy to incisional biopsy in providing satisfactory preoperative diagnoses of malignant tumors in salivary glands. Key words:Neoplasms, salivary glands, fine needle aspiration biopsy.

Purpose: Eosinophils are multifunctional leukocytes with unknown mechanisms in salivary gland tumors. Some researchers associate eosinophils with tumor progression and others have proposed them as antitumoral. The present study was conducted to compare the density of eosinophils in common salivary gland tumors and its relationship with the degree of differentiation in malignant salivary gland tumors. Materials and Methods: In this present descriptive-analytical, cross-sectional study, 15 cases of pleomorphic adenoma, 15 cases of adenoid cystic carcinoma (ACC), 30 cases of mucoepidermoid carcinoma (MEC), and 5 cases of normal salivary glands were extracted. Sections were prepared of these cases for Congo red staining. The malignant salivary gland tumors were classified as high-grade and low-grade malignancies. The slides were observed under ×10, and the highest-density areas were selected and counted in ten microscopic fields under ×40. Results: The density of the eosinophils was 4.5 ± 5.6 in the pleomorphic adenoma, 16.2 ± 6.01 in the low-grade MEC, 1.05 ± 1.1 in the high-grade MEC (Grade III), and the ACC, and 0.4 ± 0.89 in the normal salivary gland. Eosinophil density was significantly higher in low-grade malignancies compared to in benign or high-grade malignant neoplasms and normal salivary gland (P &lt; 0.001). Conclusion: This is suggested which the density of eosinophils is associated with the process of tumorigenesis and the degree of malignancy in malignant salivary gland tumors.