Biobanking of Salivary Gland Tumours: Insights from the Polish Salivary Gland Network Towards Future Digital Integration and Image-Based Research.

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.

Despite its rarity, information on the diagnosis of parapharyngeal space tumors such as through imaging and aspiration biopsy cytology, is slowly accumulating. Little detailed examination has been conducted, however, on surgical approach, complications, and sequelae. We report the results of a retrospective review of 27 patients with primary parapharyngeal space tumors-25 with benign disease and 2 with malignant lesions-treated surgically. Surgical approach, postoperative complications, sequelae, and operative indications of parapharyngeal space tumors were examined in 28 operations on the 27 patients. Tumors found in the prestyloid region in CT or MRI are treated as salivary gland or malignant tumors. Those found in the poststyloid region are treated as shwannoma or paraganglioma. The transcervical approach is often used in patients with shwannoma, while a variety of approaches are selected for patients with salivary gland tumors. Complications occur in 50% of patients, however, bias based on pathological diagnosis has not been examined to the degree needed. Sequelae in our series occurred in 46.4% of our patients. Sequela in the patients with shwannoma, however, is 81.8%, compared to 9.1% in patients with salivary gland tumors. Prestyloid parapharyngeal space tumors seem to be "automatically" indicated for surgery, because the surgical risk is lower than the risk of inaction. In poststyloid parapharyngeal space tumors, however, it appears necessary to judge indication for surgery more carefully while considering the social background, age, and occupation of prospective surgical candidates.

Salivary gland (SG) neoplasms (SGNs) display considerable immunophenotypic diversity. A significant proportion of SG carcinomas develop metastases, with increased diagnostic difficulty at metastatic sites. Transcriptional repressor GATA binding 1 (TRPS1), a novel immunohistochemical marker for breast cancer, has been found to stain certain SGNs. To investigate TRPS1 and SRY-related HMG-box 10 (SOX10) immunoexpression in various SGNs and non-SG carcinomas, head and neck paragangliomas, and head and neck mucosal melanomas. TRPS1 immunoreactivity score (IRS) was determined as negative or low, intermediate, or high positive; SOX10 was reported as negative or positive. One hundred forty-eight SGNs, 5 breast carcinomas, 105 nonbreast-non-SG carcinomas, including 33 head and neck squamous cell carcinomas (HNSCCs), 6 head and neck paragangliomas, and 6 head and neck mucosal melanomas, were assessed for TRPS1. All 23 benign SGNs showed TRPS1 positivity, with the majority having high-positive IRS (17 of 23 cases; 74%). Among 125 SG carcinomas, 115 of 125 (92%) were TRPS1 positive, with high-positive IRS in 94 of 125 (75%), intermediate-positive IRS in 15 of 125 (12%), and low-positive IRS in 6 of 125 (5%). Among nonbreast-non-SG carcinomas, HNSCC, lung, thyroid, kidney, and ovarian carcinomas showed frequent TRPS1 staining. Nearly half of HNSCCs had high (11 of 18; 33%) or intermediate (4 of 18; 12%) positive IRS. Mean IRS in SG carcinomas was significantly higher than that in nonbreast-non-SG carcinomas (P < .001). None of the TRPS1-positive nonbreast-non-SG carcinomas expressed SOX10. TRPS1 is positive in most benign and malignant SGNs. Its expression in several nonbreast-non-SG carcinomas indicates that it lacks specificity for breast and SG carcinomas, even if considering only high-positive IRS. Addition of SOX10 can increase the discriminatory utility of TRPS1.

3025 Background: Human epidermal growth factor receptor 2 ( HER2, ERBB2) amplification occurs in 5% of non-breast non-gastric solid tumors. Ado-trastuzumab emtansine (T-DM1) showed preliminary efficacy in patients with HER2 amplified lung, endometrial, salivary gland, biliary tract and ovarian cancers, but the extent of its differential effects across histologies is unknown. Methods: Patients with HER2 amplified solid tumors were enrolled and received treatment 3.6mg/kg IV every 3 weeks. The primary endpoint was overall response rate (ORR) using RECIST v1.1 or PERCIST. A basket trial expansion used a Simon two stage optimal design applied to each of 5 histology cohorts with type I error rate under 2%, power of 90%, H0 10%, H1 40%, with family-wise error rate at 10%. After first stage, lung, salivary gland and endometrial cohorts were expanded to 23 patients. The null hypothesis was rejected for each cohort separately, if at least 6 responses were observed in each cohort. Secondary endpoints included duration of response (DOR), progression-free survival (PFS) and toxicity. HER2 amplification was identified by fluorescence in situ hybridization (FISH), or next generation sequencing (NGS). Correlative studies were performed using tissue immunohistochemistry (IHC). Plasma cell-free DNA (cfDNA) was collected throughout study treatment. Results: 88 patients with 8 unique cancer types were treated across 5 cohorts of HER2 amplified lung, salivary gland, colorectal, endometrial and other cancers. The median age was 66 (26-90). Median line of prior therapy was 2 (1-7). ORR was 33% (29/87 including 11 CRs, 95% CI 24-44%), including 47% (9/19) for lung cancers, 87% (13/15, 8 CRs) for salivary gland cancers, 22% (5/23, 3 CRs) for endometrial cancers, 12% (1/8) for biliary cancers, 14% (1/7) for ovarian cancers. Median DOR was 9.7 months (95% CI 4.8, 20.2), median PFS was 2.76 months (95% CI 2.53, 5.39). There were 8 (9%) G3 treatment related toxicities. HER2 fold change corrected for purity and ploidy by FACETS algorithm correlated with response (p=0.04) and PFS&gt;=3 months (p=0.0037). HER2 amplification by NGS correlated with HER2/CEP17≥2 by FISH (67/71 tested) and IHC3+ (54/68 tested). We observed persistent HER2 amplification in plasma cfDNA during acquired resistance and progression on T-DM1 in patients with salivary gland cancers. Conclusions: Ado-trastuzumab emtansine showed promising efficacy in patients with HER2 amplified lung and salivary gland cancers as identified by NGS, meeting the primary endpoint. However, its efficacy did not meet prespecified response rate in patients with HER2 amplified endometrial, colorectal and other cancers. Histologic lineage differences in HER2 amplified cancers affect response and translational research is critical for further drug development. Clinical trial information: NCT02675829 .

Objective To evaluate the CT and MR imaging findings of salivary duct carcinoma (SDC) in order to enhance the understanding of this rare disease.Methods A retrospective analysis of CT and MRI images was performed in 20 patients (14 males and 6 females,median age 56 years old) with pathologically proved SDC.CT and MR images were evaluated with respect to the following feature factors:location,size,morphology,margin,CT density/ MR signal intensity and enhancement pattern.Results Thirteen lesions were located in parotid gland,4 lesions in submandibular gland,1 lesion in sublingual gland and 2 lesions in the buccal spaces.The maximum diameter of SDC ranged from 1.5 to 7.0 cm,mean (3.5 ±0.9) cm.Five cases demonstrated round or oval round masses with well-defined margin,15 cases demonstrated irregular masses with ill-defined edge.Among them,the peritumoral fat tissues were infiltrated in 7 cases,the preauricular skin were invaded in 3 cases,the jaw muscles were invaded in 2 cases and the parapharyngeal space,pterygoid muscle,retromandibular vein was invaded in 1 case respectively.Lesions were homogeneous in 4 cases and heterogeneous in 16 cases.Seven lesions showed varied calcifications.Lesions were homogeneous iso-intense in 1 case and heterogeneous iso-intense in 2 cases on T1WI,heterogeneous hyperintense in all cases on T2WI.On post contrast images,lesions demonstrated remarkable enhancement in 17 cases,moderate enhancement in 3 cases.Enlarged cervical lymph nodes were found in 12 cases.Conclusions SDC has nonspecific imaging characteristics.CT and MR examinations can accurately demonstrate the extent of tumor involvement and are helpful to provide more comprehensive information for SDC management. Key words: Salivary gland neoplasm; Salivary ducts; Tomography, X-ray computed; Magnetic resonance imaging

Introduction: Salivary gland tumors are common neoplasms encountered in the head and neck region. Salivary gland neoplasms have varied histopathological findings with some of the tumors showing morphologic overlap. Majority of these neoplasms are benign in nature. They occur in both major and minor salivary glands. Benign tumors are more common in major salivary glands and malignant tumors generally occur in minor salivary glands. 80% of major salivary gland tumors occur in the parotid gland. Most of the minor salivary gland tumors are located in the palate. Proper diagnosis of malignant tumors with accurate staging is very important in the treatment and management of the patient. Aims and Objectives: This study was carried out to study the histopathological spectrum of salivary gland neoplasms in a tertiary care center.Materials and methods: The present study was conducted in the Pathology department of M.S Ramaiah Hospital on the surgically resected salivary gland tumor specimens received for routine histopathological evaluation, from January 2010 to December 2014. A total of 66 cases of salivary gland neoplasms were included in the study.Results: Out of total 66 cases studied, 38 cases (57%) were benign and 28 cases (43%) were malignant. Most common age group for salivary gland tumor was between 40- 60 years. Among the tumors, female preponderance was seen in all except Warthin tumor. Pleomorphic adenoma was the most common benign tumor and mucoepidermoid carcinoma was the common malignant tumor. Parotid gland was the commonest site for various tumors, notable exception being adenoid cystic carcinoma, which showed predilection for the minor salivary glands. Pleomorphic adenoma was the most common salivary gland tumor representing 76.3% of benign and 43.9% of total salivary gland neoplasmsConclusion: Due to the varied histopathological findings with some morphologic overlap in salivary gland neoplasms histopathological examination is the mainstay for diagnosis and clinical management. Pleomorphic adenoma is the most common neoplasm of salivary glands.Benign tumors are more common in major salivary glands and malignant tumors are more common in minor salivary glands.

Over the past few decades, salivary gland tumor pathology has evolved. This includes recognition of newly defined entities as well as reclassification of other salivary gland tumors [1]. The development of genetic tests have shown that some salivary gland tumors have genetic abnormalities which are specific to a histologic type such as MECT1–MAML2 gene fusion in mucoepidermoid carcinoma and PLAG1 or HMGA2 gene translocation in pleomorphic adenoma. Immunohistochemical studies have aided in both the diagnosis and prognosis of salivary gland tumors. High Ki67 is correlated with poor overall survival in mucoepidermoid carcinoma, acinic cell carcinoma and adenoid cystic carcinoma. High MUC1 in mucoepidermoid carcinoma is associated with higher grade and high recurrence while MUC4 is associated with a lower grade tumor and longer disease free survival. The finding of androgen receptors in salivary duct carcinoma has led to new therapies as these tumors are shown to be responsive to androgen deprivation therapy. Newly described salivary gland entities in the past 20 years include both benign (sclerosing polycystic adenosis, sialolipoma) and malignant (cribriform adenocarcinoma of the tongue, mammary analogue secretory carcinoma) tumors [2]. Newly recognized histologic variants of well-known salivary gland tumors have been reported, to include salivary-duct carcinoma, acinic cell carcinoma and epi-myoepithelial carcinoma [1, 2]. In view of these dramatic changes, the Head and Neck Pathology journal leadership are particularly pleased to bring together an international panel of experts in salivary gland pathology as contributors to this special issue. Guest editors, Michal Michal, M.D, Ph.D and Alena Skalova, M.D, Ph.D have developed a special issue on salivary gland pathology that encompasses the advances in classification, genetics and other emerging concepts in the field. This update on salivary gland pathology is divided into four groups: tumors associated with molecular genetics; high grade and low grade salivary gland carcinomas; histologic variants of benign and malignant salivary gland tumors; and non-neoplastic lesions. Dr. Michal and colleagues first reported on cribriform adenocarcinoma of the tongue in 1999 and since then other pathologists have recognized this rare entity not only in the tongue but also in other minor salivary gland locations. Dr. Michal describes the characterization of this tumor including immunohistochemical and molecular studies. Dr. Goran discusses the salivary gland neoplasms that have fusion oncogenes and the clinical impact of these findings. Dr. Weinreb focuses on hylanizing clear cell carcinoma and in particular the EWSR1-ATF1 fusion set within a discussion of the differential diagnosis. Dr. Skalova rounds out the section on molecular-defined tumors with the features of mammary analogue secretory carcinoma, originally described by Dr. Skalova in 2010. Dr. Nagao discusses high grade transformation and dedifferentiation in both adenoid cystic carcinoma and acinic cell carcinoma. Newly recognized histologic variants of the high grade salivary duct carcinoma such as mucin-rich variant and sarcomatoid variant are described by Dr. Simpson. In contrast to this high grade salivary gland malignancy, Dr. Perez-Ordonez discusses low-grade salivary duct carcinoma and other intraductal carcinomas. Dr. DiPalma provides a review of carcinoma ex pleomorphic adenoma with special emphasis on the evolution of this neoplasm with distinction of the in situ category from minimally invasive and frankly invasive tumors. Recent studies have broadened the histologic range of epithelial-myoepithelial carcinoma and Dr. Seethala describes some of the new variants of this tumor. Dr. Gnepp reviews unusual mucinous tumors of salivary glands and Dr. Agaimy focuses on fat-containing salivary gland neoplasms. Dr. Petersson discusses a rare salivary gland entity: sclerosing polycystic adenosis, first characterized in 1996. We hope these stimulating discussions on common and rare salivary gland lesions will provide our readership with new tools to enable improved diagnosis.

Introduction: Salivary gland tumors are rare head and neck lesions. The majority are benign, with only 20% of cases malignant. An epithelial-myoepithelial carcinoma is a rare low-grade malignant salivary and lacrimal gland tumor, which accounts for less than 1% of salivary gland tumors. It mostly involves the parotid gland and affects adults in their sixth and seventh decades. It is usually painless, unless the minor salivary glands are affected. Case Presentation: We describe a rare case of a 35-year-old woman who presented with a 1-year-old unilateral painful parotid swelling in December 2014. Conclusions: A complete immunohistochemical study should be considered in cases of malignant salivary gland tumors. To reduce the recurrence rate of these tumors, the optimum therapies are radiotherapy and chemotherapy.

Immunohistochemical expression of the oncogenic molecules active Stat3 and survivin in benign and malignant salivary gland tumors

Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including Adenoid cystic carcinoma (ACC), Acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In this study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, total 13,643 salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2, and myoepithelial cells/stromal cells, with differentially expressed genes of each group. The localization of each cell group was anatomically validated by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Gene ontology analysis for this group included “epithelial cell differentiation” confirming the intercalated duct. Furthermore, to elucidate the potential etiologic relationship between normal salivary glands cell type and salivary gland carcinoma histology, we compared the expression of each cluster marker to SGC public RNA-seq data. In comparison between AciCC and ACC, we used normalized RNA-seq data of 54 ACC and 21 AciCC. ACC had significantly less expression of acinar cells markers (STATH, CST2, and LPO) compared to other markers expression(***P = 4 × 10−51), unlike AciCC (P = 0.54), suggesting a possible contribution of acinar cells to the carcinogenesis of AciCC. In another cohort, we compared 16 SDC, and 38 MECA with publicly available RNA-seq data. Remarkably, HES1 and ELF3, each of two different ductal cell markers, were significantly upregulated in SDC consistent with origin of SDC from ductal cells (P=0.001, P=0.0007, respectively). Consistent with bulk RNA-seq results, expression of HES1 in IHC using clinical human SDC and MECA samples was higher in SDC than MECA, suggesting that the expression of the derived cells is involved in salivary gland carcinogenesis. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship. On the other hand, there was no significant difference in myoepithelial marker ACTA2 expression between SDC and MECA, suggesting that the markers of origin are not always highly expressed, depending on cancer subtypes. Although publicly available SGC RNA-seq data used in this study are limited, the results remarkably reflect the diverse modes of carcinogenesis of SGC. These findings might provide a better understanding of the molecular basis of SGC and treatment strategies for SGC. Citation Format: Takuya Nakagawa, Chanond Nasamran, Jingjing Hu, Prakriti Sen, Theresa Guo, Kathleen Fisch, Joseph Califano. Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1076.

Introduction: Salivary gland shows various pathological conditions ranging from cystic, inflammatory, tumor like and neoplastic lesions. Fine needle aspiration cytology (FNAC) plays an important role in evaluating salivary gland (SG) tumors. Salivary gland tumors are one of the most heterogeneous groups of neoplasms with cytopathological features overlapping among the entities and making it difficult to assign to specific category. Due to these facts, salivary gland cytopathology is one of the most challenging areas of cytology. The lack of a uniform reported guidelines in salivary gland cytopathology leads to inter-observer variability and disagreements. The present study was undertaken to assess the degree of inter-observer reproducibility for diagnostic categorization of salivary gland lesions utilizing MSRSGC among pathologists with varying experience along with its role in providing a framework for reporting salivary gland lesions. Materials and Methods: In this cross sectional study, total of 44 cases of salivary gland lesions subjected to FNAC over a period of 7 year were studied. The cases were critically reviewed by 2 pathologists and a pathology resident with variable experience in cytopathology using MSRSGC in our institution. Inter-observer variability was assessed by comparing the agreement between two cytopathologists and pathology resident by using Cohen’s kappa statistics (Io score) and interpretation of the results was done using scale of Landis and Koch. Results: All the salivary gland aspirates were categorized according to MSRSGC.Out of 44 cases, maximum cases 22 (50%) were classified under IVA (BN) followed by 27.27% to 29.5% cases classified under II (NN), 2.27-4.55% of cases under Category IVB (SUMP), 4.55% under category V( SM) and 6.82% cases under category VI ( M). Inter-observer variability (IOV) was calculated for individual category in Milan system using Cohens kappa test, which was found to be in the almost perfect agreement range as per Landis and Koch, for categories II, IVA, V, VI ( Io score 0.89- 1). Kappa score ranged from 0.645 - 1 for category I (ND), which showed substantial to an almost perfect agreement. Whereas, category IVB (SUMP) showed variable results, with substantial agreement (Io score 0.656) to no agreement (Io score 0) between different observers.The overall IOV showed an almost perfect agreement with a kappa score of 0.861(obs1 vs 2) ,0.896 (obs1 vs R), 0.965 (obs 2 vs R). The data was found to be statistically significant (p=< .0001). Conclusion: MSRSGC is a very efficient system and has the potential to standardize salivary gland FNA diagnoses, providing clear prognostic and management information to clinicians and surgeons. This system can be used with good reproducibility between observers with variable cytopathology experience. Placing lesions in categories using MSRSGC can result in optimal management of discordant cases without using a specific diagnosis. Application of MSRSGC has immense value for standardization of reporting of salivary gland FNAC.Hence we recommend the use of Milan system for reporting salivary gland cytopathology.

Parapharyngeal space tumours (PPS) are benign, uncommon located in the head and neck region. Pleomorphic adenoma the most prevalent type of salivary gland tumour can develop from the salivary glands or start in the deep lobe of the parotid gland and spread into PPS. A 59-year-old male presented with complaints of dysphagia, voice change and swelling in the soft palate. PPS tumour was detected with endoscopy, biopsy confirmed it as salivary gland tumour and PET-CT scan findings revealed a large hyper metabolic solid soft tissue density lesion in left parapharyngeal space with local extent. Tumour excision was done through transcervical approach. Biphasic components were highlighted by CK7, SOX10, SMA, P63 using immunohistochemistry (IHC) confirming benign mixed salivary gland tumor pleomorphic adenoma. Patient was treated with IV antibiotics, IV PPI’s, IV analgesics, IV antiemetics and discharged in stable condition. PET-CT, IHC serves a vital role in diagnosis and confirmation of severity of tumour. The transcervical approach is the preferred technique for excising tumours in the parapharyngeal space that have narrow attachments.

Nearly half of parapharyngeal space (PPS) tumors present as an intraoral mass, which is diagnostically challenging. In this study, we studied whether preoperative growth patterns were associated with histopathological diagnosis for planning surgery. We performed a cross-sectional study in patients with PPS tumors. A simplified classification scheme based on intraoral tumor growth patterns (patterns 1 and 2) was then proposed. In pattern 1, tumors bulge submucosally to the oropharynx from the soft palate, with the center convexity above the uvula. In pattern 2, tumors bulge submucosally to the oropharynx from the lateral oropharynx wall, with the center convexity below the uvula. The association of this classification with postoperative histopathological diagnosis and surgical-related events was studied. Twenty-two patients were enrolled in this study (12 with pattern 1, 10 with pattern 2). Of these, 91.7% (11/12) of pattern 1 tumors were salivary gland tumors (P < .001), and 90% (9/10) of pattern 2 tumors were neurogenic (P < .001). Pattern 2 tumors had fewer bleeding complications or needed external approaches when a transoral approach was chosen. This new classification of PPS tumors facilitates the prediction of salivary gland and neurogenic tumors and can improve the accuracy of preoperative radiologic diagnosis. This system will be helpful for planning surgical interventions, such as implementing transoral approaches.

One of the biggest challenges in managing head and neck cancers, especially salivary gland cancers, is the identification of secreted biomarkers of the disease that can be evaluated noninvasively. A relevant source of enriched tumor markers could potentially be found in the tumor secretome. Although numerous studies have evaluated secretomes from various cancers, the influence of the cancer secretome derived from salivary gland cancers on the behavior of normal cells has not yet been elucidated. Our data indicate that secretome derived from salivary gland cancer cells can influence the expression of two potential biomarkers of oral cancer-namely, bone sialoprotein (BSP) and dentin sialoprotein (DSP)-in normal salivary gland cells. Using routine immunohistochemistry, immunofluorescence, and immunoblotting techniques, we demonstrate an enrichment of BSP and DSP in human salivary gland (HSG) cancer tissue, unique localizations of BSP and DSP in HSG cancer cells, and enriched expression of BSP and DSP in normal salivary gland cells exposed to a cancer secretome. The secretome domain of the cancer microenvironment could alter signaling cascades responsible for normal cell proliferation, migration, and invasion, thus enhancing cancer cell survival and the potential for cancer progression. The cancer secretome may be critical in maintaining and stimulating "cancer-ness," thus potentially promoting specific hallmarks of metastasis.