APC-related multiple salivary gland lesions: spatial transcriptomic analysis reveals progressive WNT activation

APC-related multiple salivary gland lesions: spatial transcriptomic analysis reveals progressive WNT activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report APC -related multiple salivary gland lesions: spatial transcriptomic analysis reveals progressive WNT activation Fiona Chan-Pak-Choon, Catherine Beaumont, José Camacho Valenzuela, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6557806/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Attenuated familial adenomatous polyposis (AFAP) is a disorder caused by germline pathogenic variants in APC and is characterized by the presence of <100 colonic polyps and a high lifetime risk of developing colorectal cancer. Salivary gland basal cell tumours are uncommon and have not been reported in AFAP before. We present a family with AFAP and multiple salivary gland tumours, including basal cell adenoma (BCA) and basal cell adenocarcinoma (BCAC). The colon and salivary gland tumours showed abnormal nuclear beta-catenin staining. Genomic analysis of both parotid BCACs showed CNN-LOH at the APC locus, implicating loss of full-length APC in the aetiology of the parotid BCACs. In contrast, the submandibular BCAC showed a p.(Ile35Thr) CTNNB1 mutation. Spatial transcriptomic analysis revealed a stepwise increase in the expression of WNT pathway genes across the proband’s salivary lesions, from benign (intercalated duct hyperplasia and BCA) to malignant (BCACs). Our results showcase BCA and BCAC as new phenotypes of AFAP. Cancer Biology Molecular Genetics Medical Genetics Familial adenomatous polyposis APC WNT pathway Basal cell adenocarcinoma Basal cell adenoma spatial transcriptomics Figures Figure 1 Figure 2 Figure 3 Introduction Salivary gland tumours are uncommon, with an incidence of 0.5-2 per 100,000 individuals and accounting for 6% of all head and neck tumours, of which 20% are malignant [1]. Common salivary neoplasms include pleomorphic adenomas (65% of benign entities), mucoepidermoid and adenoid cystic carcinomas (33% and 24% of malignancies, respectively) [2]. In contrast, basal cell adenomas (BCAs) and adenocarcinomas (BCACs) represent only 5-6% of benign and 1-3% of malignant salivary tumours, respectively [3]. These tumours harbour somatic CTNNB1 mutations, leading to abnormal nuclear b-catenin (encoded by CTNNB1 ) accumulation and overactivation of WNT signalling target genes that regulate cellular processes such as transcription, cell cycle control and apoptosis [3,4]. As far as we are aware, no cases of BCA or BCAC have been reported in association with a germline pathogenic variant (PV) in the colorectal cancer predisposition gene, APC , although one case of BCA has been reported to harbour two APC missense alterations of questionable significance [5]. Attenuated familial adenomatous polyposis (AFAP) is a hereditary disorder characterized by the development of 100 colonic polyps and an almost certain progression to CRC by age 35-40y if no prophylactic colectomy is carried out [4]. Both FAP and AFAP are caused by germline PVs in APC , which encodes APC, a key member of the WNT pathway [4]. Extracolonic manifestations of FAP/AFAP include osteomas, desmoid tumours and duodenal polyps, among others [4]. In contrast, salivary gland tumours in FAP/AFAP are very rare, with only three reported cases: a pleomorphic adenoma, fibromatous tumour and mucoepidermoid carcinoma [6-8]. Here, we describe the first familial case of AFAP with multiple BCAs and BCACs. We confirm the involvement of APC and CTNNB1 by sequencing the tumours of the three affected family members. To further explore this novel association, we carry out spatial transcriptomic analyses and show a progressive increase in WNT genes expression levels from intercalated duct hyperplasia (IDH) to BCA to BCAC. Materials and methods Patient samples Blood from the proband and formalin-fixed, paraffin-embedded (FFPE) or fresh frozen tumours from the proband, his sister and mother were collected – proband’s colon polyps, bilateral parotid BCACs, submandibular BCAC, papillary thyroid carcinoma (PTC), the sister’s BCA and the mother’s colon polyps and CRC. DNA was extracted as described previously [9]. Immunohistochemistry (IHC) The tumours were stained by IHC using b-catenin (Clone 14, Roche #05269016001, prediluted). Whole exome sequencing (WES), panel sequencing and copy-number variant (CNV) analyses WES data from the germline and tumour DNA were processed for single-nucleotide variant (SNV) and CNV analyses as described previously [10]. Panel sequencing and additional CNV analyses are described in the supplementary file. Digital spatial profiling (DSP) Four-mm sections from three FFPE blocks containing the proband’s BCACs (parotid and submandibular), BCA, IDH and normal salivary tissue were mounted on a single glass slide. DSP was carried out using the NanoString GeoMx DSP platform and the Whole Transcriptome Atlas RNA panel. Pan-cytokeratin (PanCK) (Clones AE1+AE3, Novus Biologicals #NBP2-33200AF532, 1:75) and DNA stain (SYTO13, Thermo Fisher Scientific #S7575, 500nM) were used to select 28 regions of interest (ROIs) by immunofluorescence. Within the ROIs, 28 PanCK+ areas of illumination (AOIs) were selected in each tissue subtype (Figure 2). A mask for 9 PanCK- AOIs was also selected (data not shown). RNA data was filtered, normalised, and analysed on the GeoMx DSP data analysis platform (GeomxTools 3.10.0 and GeoMxWorkflows 1.12.0) [11]. Gene set enrichment analysis (GSEA) was performed using GSEA (Broad Institute version 4.3.3) and MSigDB (version MSigDB 2024.1.Hs) [12]. Results The affected family members – the proband, his sister and their mother – carried a likely pathogenic germline deletion in APC , (NM_0000038.6):c.4910_4923del, p.(Asp1637Valfs*4) [PVS1, PM2_supporting [13]], and have a history of colon polyps (n<40) (Figure 1A). The proband, a 40-year-old male, presented with multiple bilateral parotid gland masses (BCA and BCACs) and subsequently developed a submandibular gland BCAC at age 41y and a PTC at 42y. The sister developed a parotid BCA at 38y, and the mother died of CRC at 60y. There were several lesion subtypes in the proband’s bilateral parotid tumours – IDH (unencapsulated; benign), BCA (encapsulated; benign) and BCAC (encapsulated with capsular invasion; malignant) (Figure 1B). However, the submandibular tumour consisted of BCAC only (Figure 1B) and the sister’s parotid mass of BCA only. Abnormal b-cateninnuclear staining by IHC was observed in the abluminal cells of all the family’s tumours except for the proband’s PTC (Figure 1B; Table 1). WES and/or panel sequencing confirmed the germline APC deletion in the proband’s blood, and tumours developed by the family (Table 1). Somatic truncating APC mutations (second/third hits) were found in the proband’s colon polyp and the mother’s colon polyp and CRC. TP53 mutation, (NM_000546.6):c.713G>A, p.(Cys238Tyr), was also found in the mother’s CRC (Table 1). In the proband’s bilateral parotid tumours, copy-number neutral loss of heterozygosity (LOH) at the APC locus, extending to ~120Mb of chromosome 5q (Chr5q) was identified. However, the submandibular BCAC showed no Chr5q LOH (Figure 1 C-D; Figure S1) and instead carried a somatic CTNNB1 mutation, (NM_001904.4):c.104T>C, p.(Ile35Thr) at 48.2%. Truncating APC mutations were also found in the proband’s and sister’s BCAs, but not in the proband’s IDH. Additionally, BRAF mutation, (NM_004333.6):c.1799T>A, p.(Val600Glu) was detected in the proband’s PTC (Table 1). To further examine the relationship between the different salivary gland tumour subtypes and their expression profiles, spatial transcriptomic analyses were carried out on the proband’s lesions. Pan-CK+ AOIs in the IDH, BCA, parotid BCAC, submandibular BCAC and normal salivary gland tissue were selected for the analysis (Figure 2). UMAP analysis revealed two clusters, benign and malignant tissues, based on the expression of 16,489 genes (Figure S2). GSEA consistently identified the WNT signalling pathway among the top enriched gene-sets when comparing the tumour subtypes and normal salivary tissue (Figure S3). Subsequently, an unsupervised hierarchical clustering analysis using the expression levels of the main WNT genes only (n=89) [14] yielded five distinct clusters, one per tumour/tissue subtype, with the two main branches separating the malignant and benign tissues. CTNNB1 expression levels were significantly different (t-test, p<0.05) between each subgroup, apart from the BCA and IDH, whereas APC expression levels were similar across all subtypes (Figure 3). WNT-GSEA scores were used to quantify the WNT activation associated to each tumour subtype (from IDH to BCA to BCACs) and their molecular background. There was an overall increase in WNT activation from IDH [no CTNNB1 p.(Ile35Thr] or second hit in APC ) to BCA [ APC c.1161_1162del, p.(His388Glnfs*8)] to parotid BCAC (Chr5q LOH) to submandibular BCAC [ CTNNB1 p.(Ile35Thr)] (Figure S4). Discussion BCAs and BCACs are morphologically similar, with the only distinction being the invasive tumour growth beyond the capsule in BCACs [15]. Malignant transformation of BCA to BCAC has been proposed, analogous to the suggested role of IDH as a precursor to BCA [16,17]. The nuclear b-catenin staining seen in IDH, BCAs and BCACs indicates WNT pathway involvement. However, activating CTNNB1 mutations have primarily been observed in BCAs (50%; n=88/176) but only occasionally in BCACs (13%; n =4/30) and IDH (20%; n=3/15) (Tables S1-S2) [5,15,17-23]. Recently, FBXW11 , also a WNT family gene, was identified as a new driver in BCAs lacking CTNNB1 variants [23]. In a limited number of BCACs, variants in CYLD , PIK3CA and HRAS have been reported, but not in a WNT gene other than CTNNB1 or FBXW11 [3,15,20,23]. In the familial case presented herein, a germline deletion in APC and somatic mutations in either APC or CTNNB1 were identified in the salivary and colon tumours, partially fitting the b-catenin/WNT signalling model (Table 1). The APC domain involved in b-catenin binding and degradation consists of seven (14 with both alleles) 20-amino-acid repeats (20-AARs) [24]. In FAP/AFAP tumours, APC PVs typically result in a truncated protein retaining one to three 20-AARs, encoded by codon 1250-1450 and known as the mutation cluster region. This ensures the ‘just-right’ amount of β-catenin regulation is maintained, promoting an optimal level of WNT activity for tumorigenesis [24,25]. In the patients described, only the proband’s BCA and mother’s colon polyp followed this ‘just-right’ model, by retaining three 20-AARs, whereas the other tumours retained four to ten 20-AARs (Figure 1C, Table S3). Histopathological studies have indicated a possible progression from BCA to BCAC [16,26,27]. Although the numbers of cases are limited, the aberrations in genes affecting different signalling pathways in BCAC and BCA suggest that most BCACs likely arise de novo and not from BCA, although there is some overlap and more studies are required [15,20,23]. In our proband, the absence of shared somatic variants among the salivary tumours and the distinct second hits detected in each lesion suggest that they developed independently and thus, the BCACs did not arise from the BCA (Figure 1C; Table 1). Rather, multiple primary tumours at different stages of tumorigenesis were observed, which is in-line with the predisposition to multiple cancers caused by a germline APC deletion. Spatial transcriptomic analysis of the proband’s salivary gland lesions and normal tissue showed distinct clustering based on subtype, with WNT scores progressing from benign to malignant tissues (Figure 3; Figure S4). The increase in WNT scores reflects the gradient in aggressivity expected from benign lesion (IDH) to benign tumour (BCA) to malignant tumours (BCACs). Interestingly, the submandibular BCAC with the somatic CTNNB1 mutation, p.(Ile35Thr), had a higher WNT score than the parotid BCAC with Chr5q LOH. Remarkably, the CTNNB1 p.(Ile35Thr) mutation is almost exclusively seen in salivary gland tumours (94%; n=72/77) (c-square test, p<10 -15 ) (Table S4-5) [5,15,17-23,28,29]. This suggests that the CTNNB1 p.(Ile35Thr) mutation somatically can lead to BCAC development and has a greater impact on WNT dysregulation than does APC inactivation, possibly due to its high specificity to salivary gland tumours. In summary, spatial transcriptomic analysis revealed a stepwise increase in WNT activation from IDH to BCA to BCAC. Additionally, the proband’s BCACs did not appear to arise from the BCA. Our findings showcase salivary gland tumours, BCA and BCAC, as new phenotypes of AFAP, highlighting that more than 30 years since the discovery of APC as the cause of FAP, new phenotypes are still being uncovered. Declarations Acknowledgements : We would like to thank Tamiko Nishimura, Naciba Benlimame and Lilian Canetti for their support in the spatial transcriptomic assay. We thank Anne-Sophie Chong for her help sequencing the PTC and Jesús del Valle for his assistance in evaluating the pathogenicity of the germline APC deletion. We acknowledge the molecular genetics laboratory team of the Institute of Human Genetics in Ulm for their help with the OncoScan and CytoScan assays. This study was funded by the Canadian Institutes of Health Research grant (FDN-148390) to WDF. BR is a Miguel Servet Fellow (CP21/00038) from the Instituto de Salud Carlos III (PI20/01721). FCPC is supported by the George G Harris Fellowship. JCV was supported in part by an internal award from The Research Institute of the McGill University Centre (RI-MUHC). This research as well as biobanking of biological material and data was made possible in part through a collaboration with the Réseau de recherche sur le cancer (RRCancer) financially supported by the Oncopole, the FRQ cancer division, which receives funding from Merck Canada Inc., GSK, Pfizer and the Ministère de l'Économie, de l'Innovation et de l'Énergie du Québec. The RRCancer is affiliated to the Canadian Tumor Repository Network (CTRNet). Author contributions : FCPC collected the samples, carried out the genomic and spatial transcriptomic experiments and analyses and drafted the manuscript. CB, FT, JL and TCM provided the patient samples and clinical information. SD and RS conducted the OncoScan and CytoScan analyses. MP and JC performed pathology reviews and participated in the spatial transcriptomic assay. JCV and PP assisted with the genomic analyses. BR and WDF oversaw all aspects of the project and edited the manuscript. All authors approved the submission of the manuscript. Patient consent statement : Written informed consent was obtained from all patients involved in the study. Data availability : The data supporting the findings of this study will be deposited in will be provided in the final version of the manuscript and made publicly available prior to publication. References To VSH, Chan JYW, Tsang RKY , et al. Review of Salivary Gland Neoplasms. International Scholarly Research Notices 2012; 2012 : 872982. Agulnik M, McGann CF, Mittal BB , et al. Management of salivary gland malignancies: current and developing therapies. Oncology Reviews 2008; 2 : 86-94. Robinson RA. Basal Cell Adenoma and Basal Cell Adenocarcinoma. Surg Pathol Clin 2021; 14 : 25-42. Galiatsatos P, Foulkes WD. 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Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/β-catenin signalling thresholds for tumourigenesis. Oncogene 2013; 32 : 4675-4682. Batsakis JG, Luna MA. Basaloid Salivary Carcinoma. Annals of Otology, Rhinology & Laryngology 1991; 100 : 785-787. Qiao C, Li D, Zhang Z , et al. Intracapsular carcinoma ex basal cell adenoma of the parotid gland. International Journal of Oral and Maxillofacial Surgery 2020; 49 : 1548-1550. Daa T, Kashima K, Kaku N , et al. Mutations in components of the Wnt signaling pathway in adenoid cystic carcinoma. Mod Pathol 2004; 17 : 1475-1482. Daa T, Kaku N, Kashima K , et al. Expression of beta-catenin, E-cadherin and cyclin D1 in adenoid cystic carcinoma of the salivary gland. J Exp Clin Cancer Res 2005; 24 : 83-87. Cameselle-Teijeiro JM, Peteiro-González D, Caneiro-Gómez J , et al. Cribriform-morular variant of thyroid carcinoma: a neoplasm with distinctive phenotype associated with the activation of the WNT/β-catenin pathway. Mod Pathol 2018; 31 : 1168-1179. Nieminen TT, Walker CJ, Olkinuora A , et al. Thyroid Carcinomas That Occur in Familial Adenomatous Polyposis Patients Recurrently Harbor Somatic Variants in APC, BRAF, and KTM2D. Thyroid 2020; 30 : 380-388. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryMaterialOnlinelegends.docx APCfinalTablesS15preprint.xlsm Supplementary Tables S1-S5 APCfinalSupplementalpreprint.pdf Online Supplementary Material Table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6557806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":449790730,"identity":"096aaaa5-beb0-4b3a-ad0e-658927f9a925","order_by":0,"name":"Fiona Chan-Pak-Choon","email":"","orcid":"https://orcid.org/0000-0002-6317-1822","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Fiona","middleName":"","lastName":"Chan-Pak-Choon","suffix":""},{"id":449791395,"identity":"5574d467-7dac-4c63-8692-740642346645","order_by":1,"name":"Catherine Beaumont","email":"","orcid":"","institution":"Department of Ophthalmology and Otolaryngology - Head and Neck Surgery, Laval University, Quebec City, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Beaumont","suffix":""},{"id":449791396,"identity":"b24dac47-fa5e-448b-8cef-cb2b9a829a38","order_by":2,"name":"José Camacho Valenzuela","email":"","orcid":"","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Camacho","lastName":"Valenzuela","suffix":""},{"id":449791397,"identity":"2d01c6f8-faee-48b2-b74d-eaa7722816d2","order_by":3,"name":"Josianne Leblanc","email":"","orcid":"","institution":"Department of Laboratory Medicine, CIUSSS Saguenay-Lac-St-Jean, Saguenay, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Josianne","middleName":"","lastName":"Leblanc","suffix":""},{"id":449791398,"identity":"ff99587f-b60b-4c0e-99c1-f67f0c2174c3","order_by":4,"name":"Sonja Dahlum","email":"","orcid":"","institution":"Institute of Human Genetics, Ulm University and Ulm University Medical Centre, Ulm, Germany","correspondingAuthor":false,"prefix":"","firstName":"Sonja","middleName":"","lastName":"Dahlum","suffix":""},{"id":449791399,"identity":"3254c476-f1f6-4371-9d54-564b8ccbf424","order_by":5,"name":"Reiner Siebert","email":"","orcid":"","institution":"Institute of Human Genetics, Ulm University and Ulm University Medical Centre, Ulm, Germany","correspondingAuthor":false,"prefix":"","firstName":"Reiner","middleName":"","lastName":"Siebert","suffix":""},{"id":449795701,"identity":"89f75fd9-c9b2-44f9-b9ae-e797d3914efa","order_by":6,"name":"François Thuot","email":"","orcid":"","institution":"Department of Ophthalmology and Otolaryngology - Head and Neck Surgery, Laval University, Quebec City, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"Thuot","suffix":""},{"id":449795702,"identity":"34ea8435-d732-4273-87a1-f71369221280","order_by":7,"name":"Marc Pusztaszeri","email":"","orcid":"","institution":"Department of Pathology, Jewish General Hospital, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Pusztaszeri","suffix":""},{"id":449795703,"identity":"5a907dcd-db23-459d-bd07-fb9cb0f3ed27","order_by":8,"name":"Jacinthe Chênevert","email":"","orcid":"","institution":"Department of Pathology, Hôtel-Dieu de Québec, CHU de Québec, Laval University, Quebec City, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Jacinthe","middleName":"","lastName":"Chênevert","suffix":""},{"id":449795704,"identity":"2e9243b9-b3d4-439b-93af-cfbfa2d6c5be","order_by":9,"name":"Paz Polak","email":"","orcid":"","institution":"Quest Diagnostics, Secaucus, NJ, USA","correspondingAuthor":false,"prefix":"","firstName":"Paz","middleName":"","lastName":"Polak","suffix":""},{"id":449795705,"identity":"461f0217-49bf-4e73-92d6-4a125d862c85","order_by":10,"name":"Tania Cruz Marino","email":"","orcid":"","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Tania","middleName":"Cruz","lastName":"Marino","suffix":""},{"id":449795706,"identity":"4061c7f7-986d-46c6-8040-3ba03092d34d","order_by":11,"name":"Barbara Rivera","email":"","orcid":"https://orcid.org/0000-0001-9434-6288","institution":"Molecular Mechanisms and Experimental Therapy in Oncology Program, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Rivera","suffix":""},{"id":449795707,"identity":"26246b63-3e1b-406c-a5ee-0385f968cee7","order_by":12,"name":"William D. Foulkes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie3RPWrDMBTA8RcM0fKgq7vYV5ApeCrkKsqUJQGPGYJRKaRLaFZfIlAvniUM9uIlW8CLTS6QKXQKlUQDocSCbqXov/gD/XiWDOBy/cU870UwdSUcRpIDBPql6BIbGXFDUABo8mQIo7YxisANmXL9ZCO0VqRbFYBkQ2W2TGe7t3qvp4QPfICU+sOqFhAbKj+aclE080STKBP3SWzIuIWJP6eyX4tFcUCmCQMruagphlzSWXwloY1M198k5x6LD0QYQgfIxJD3FhGrRGZVGam96EP2o3yAPG5L2X+e2wDJa95vVmkY1/XxdFo+h8EAuYa39/qn+Pb1PyLdr5a7XC7Xv+8L5hRtPooFCJ8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7427-4651","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"D.","lastName":"Foulkes","suffix":""}],"badges":[],"createdAt":"2025-04-29 15:18:24","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6557806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6557806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81966040,"identity":"3ddce7ff-206c-4d2d-b545-adc9a99265cd","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10725223,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree and molecular findings of AFAP family with salivary gland tumours. \u003cstrong\u003eA \u003c/strong\u003ePedigree of family. \u003cstrong\u003eB\u003c/strong\u003e H\u0026amp;E stains and b-catenin IHC of the salivary gland tumour subtypes. The basal cell adenoma (BCA) and adenocarcinomas (BCACs) are both encapsulated, but only the adenocarcinomas show capsular invasion. Abnormal b-catenin nuclear staining in abluminal cells show WNT pathway dysregulation. \u003cstrong\u003eC\u003c/strong\u003e Map of the APC and b-catenin domains with the variants identified in the family’s tumours indicated. \u003cstrong\u003eD\u003c/strong\u003eCopy-number variant profiles of the BCACs, showing copy-number neutral LOH (CNN-LOH) of Chr5q in the bilateral parotid BCACs, but not in the submandibular BCAC, where instead, there was allelic imbalance with gain of the mutated allele at Chr5q. Major allele in red, minor allele in blue and total copy number is the sum of the major and minor alleles\u003c/p\u003e","description":"","filename":"APCFig1finalpreprint.png","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/49b1b524df8d61c2be0e21b6.png"},{"id":81966042,"identity":"fc80e71d-751a-4235-b828-dc1fe03dc859","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17612930,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of GeoMx regions of interest (ROIs) and areas of illumination (AOIs) in the salivary gland tumours. The upper panel consists of images of the fluorescently stained right and left parotid and submandibular glands of the proband and the selected ROIs. The selected ROIs are demarcated by circles and rectangles that are composed of PanCK+ nuclei (in red) and/or PanCK- nuclei (in white, as a mask). In the lower panel, representative AOIs (PanCK+ only) that were sampled for RNA analysis are shown – green (PanCK+) and blue (DNA)\u003c/p\u003e","description":"","filename":"APCFig2finalpreprint.png","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/b251ec8369e35889b58038ad.png"},{"id":81966038,"identity":"ab748099-f194-46b8-8194-215a802ae6c6","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1836249,"visible":true,"origin":"","legend":"\u003cp\u003eUnsupervised hierarchical clustering of PanCK+ cells across different tissue types based on the expression of WNT genes. Each sample clusters with their respective tissue type and the main branches of the dendogram split the benign (normal salivary gland, intercalated duct hyperplasia (IDH) and BCA) from the malignant tissues (BCACs). The BCACs show a higher level of WNT genes expression compared to the other subtypes, whereas the differences between the IDH and BCA are more subtle. The red to blue gradient scale corresponds to the Z-value. The in-sets show \u003cem\u003eCTNNB1\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e normalized expression per tissue type. \u003cem\u003eCTNNB1\u003c/em\u003e expression is significantly different for all comparisons except for that of the hyperplasia and BCA. ns: not significant, *: p\u0026lt;0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"APCFig3finalpreprint.png","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/e1ac8e4f8618b4fa417c28a5.png"},{"id":81967668,"identity":"6988e57c-1355-49f0-80f9-4db30e976467","added_by":"auto","created_at":"2025-05-05 11:42:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27520559,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/7b99a02a-effa-48f0-8e4f-80596d340e62.pdf"},{"id":81966036,"identity":"77c919bf-83f6-4e74-ba9a-c21dfc8acf2d","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":132457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterialOnlinelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/8f0e1f9d0e106167393885d0.docx"},{"id":81967618,"identity":"4ec9d9a6-e53b-40de-9324-fed2a00a60e2","added_by":"auto","created_at":"2025-05-05 11:42:33","extension":"xlsm","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":36282,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Tables S1-S5\u003c/p\u003e","description":"","filename":"APCfinalTablesS15preprint.xlsm","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/78d8665a49ecba438288bb94.xlsm"},{"id":81966039,"identity":"026d432c-e7b9-49fe-98c3-c14a82cdd9af","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2059835,"visible":true,"origin":"","legend":"\u003cp\u003eOnline Supplementary Material\u003c/p\u003e","description":"","filename":"APCfinalSupplementalpreprint.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/5119741f180dd2a5c387129f.pdf"},{"id":81966035,"identity":"73de7ce8-b6e4-48b4-bff9-071872f300ee","added_by":"auto","created_at":"2025-05-05 11:34:33","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":40409,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6557806/v1/d3bd80756383614c7c979820.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAPC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-related multiple salivary gland lesions: spatial transcriptomic analysis reveals progressive WNT activation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalivary gland tumours are uncommon, with an incidence of 0.5-2 per 100,000 individuals and accounting for 6% of all head and neck tumours, of which 20% are malignant [1]. Common salivary neoplasms include pleomorphic adenomas (65% of benign entities), mucoepidermoid and adenoid cystic carcinomas (33% and 24% of malignancies, respectively) [2]. In contrast, basal cell adenomas (BCAs) and adenocarcinomas (BCACs) represent only 5-6% of benign and 1-3% of malignant salivary tumours, respectively [3]. These tumours harbour somatic \u003cem\u003eCTNNB1\u003c/em\u003e mutations, leading to abnormal nuclear b-catenin (encoded by \u003cem\u003eCTNNB1\u003c/em\u003e) accumulation and overactivation of WNT signalling target genes that regulate cellular processes such as transcription, cell cycle control and apoptosis [3,4]. As far as we are aware, no cases of BCA or BCAC have been reported in association with a germline pathogenic variant (PV) in the colorectal cancer predisposition gene, \u003cem\u003eAPC\u003c/em\u003e, although one case of BCA has been reported to harbour two \u003cem\u003eAPC\u003c/em\u003e missense alterations of questionable significance [5].\u003c/p\u003e\n\u003cp\u003eAttenuated familial adenomatous polyposis (AFAP) is a hereditary disorder characterized by the development of \u0026lt;100 colorectal adenomas (polyps) and a 69% risk of colorectal cancer (CRC) by age 80y [4]. Classic familial adenomatous polyposis (FAP) is defined by the development of \u0026gt;100 colonic polyps and an almost certain progression to CRC by age 35-40y if no prophylactic colectomy is carried out [4]. Both FAP and AFAP are caused by germline PVs in \u003cem\u003eAPC\u003c/em\u003e, which encodes APC, a key member of the WNT pathway [4]. Extracolonic manifestations of FAP/AFAP include osteomas, desmoid tumours and duodenal polyps, among others [4]. In contrast, salivary gland tumours in FAP/AFAP are very rare, with only three reported cases: a pleomorphic adenoma, fibromatous tumour and mucoepidermoid carcinoma [6-8]. \u003c/p\u003e\n\u003cp\u003eHere, we describe the first familial case of AFAP with multiple BCAs and BCACs. We confirm the involvement of \u003cem\u003eAPC\u003c/em\u003e and \u003cem\u003eCTNNB1\u003c/em\u003e by sequencing the tumours of the three affected family members. To further explore this novel association, we carry out spatial transcriptomic analyses and show a progressive increase in WNT genes expression levels from intercalated duct hyperplasia (IDH) to BCA to BCAC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePatient samples \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood from the proband and formalin-fixed, paraffin-embedded (FFPE) or fresh frozen tumours from the proband, his sister and mother were collected \u0026ndash; proband\u0026rsquo;s colon polyps, bilateral parotid BCACs, submandibular BCAC, papillary thyroid carcinoma (PTC), the sister\u0026rsquo;s BCA and the mother\u0026rsquo;s colon polyps and CRC. DNA was extracted as described previously [9]. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumours were stained by IHC using b-catenin (Clone 14, Roche #05269016001, prediluted).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole exome sequencing (WES), panel sequencing and copy-number variant (CNV) analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWES data from the germline and tumour DNA were processed for single-nucleotide variant (SNV) and CNV analyses as described previously [10]. Panel sequencing and additional CNV analyses are described in the supplementary file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDigital spatial profiling (DSP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour-mm sections from three FFPE blocks containing the proband\u0026rsquo;s BCACs (parotid and submandibular), BCA, IDH and normal salivary tissue were mounted on a single glass slide. DSP was carried out using the NanoString GeoMx DSP platform and the Whole Transcriptome Atlas RNA panel. Pan-cytokeratin (PanCK) (Clones AE1+AE3, Novus Biologicals #NBP2-33200AF532, 1:75) and DNA stain (SYTO13, Thermo Fisher Scientific #S7575, 500nM) were used to select 28 regions of interest (ROIs) by immunofluorescence. Within the ROIs, 28 PanCK+ areas of illumination (AOIs) were selected in each tissue subtype (Figure 2). A mask for 9 PanCK- AOIs was also selected (data not shown). RNA data was filtered, normalised, and analysed on the GeoMx DSP data analysis platform (GeomxTools 3.10.0 and GeoMxWorkflows 1.12.0) [11]. Gene set enrichment analysis (GSEA) was performed using GSEA (Broad Institute version 4.3.3) and MSigDB (version MSigDB 2024.1.Hs) [12]. \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe affected family members \u0026ndash; the proband, his sister and their mother \u0026ndash; carried a likely pathogenic germline deletion in \u003cem\u003eAPC\u003c/em\u003e, (NM_0000038.6):c.4910_4923del, p.(Asp1637Valfs*4) [PVS1, PM2_supporting [13]], and have a history of colon polyps (n\u0026lt;40) (Figure 1A). The proband, a 40-year-old male, presented with multiple bilateral parotid gland masses (BCA and BCACs) and subsequently developed a submandibular gland BCAC at age 41y and a PTC at 42y. The sister developed a parotid BCA at 38y, and the mother died of CRC at 60y.\u003c/p\u003e\n\n\u003cp\u003eThere were several lesion subtypes in the proband\u0026rsquo;s bilateral parotid tumours \u0026ndash; IDH (unencapsulated; benign), BCA (encapsulated; benign) and BCAC (encapsulated with capsular invasion; malignant) (Figure 1B). However, the submandibular tumour consisted of BCAC only (Figure 1B) and the sister\u0026rsquo;s parotid mass of BCA only. Abnormal b-cateninnuclear staining by IHC was observed in the abluminal cells of all the family\u0026rsquo;s tumours except for the proband\u0026rsquo;s PTC (Figure 1B; Table 1).\u003c/p\u003e\n\n\u003cp\u003eWES and/or panel sequencing confirmed the germline \u003cem\u003eAPC\u003c/em\u003e deletion in the proband\u0026rsquo;s blood, and tumours developed by the family (Table 1). Somatic truncating \u003cem\u003eAPC\u003c/em\u003e mutations (second/third hits) were found in the proband\u0026rsquo;s colon polyp and the mother\u0026rsquo;s colon polyp and CRC. \u003cem\u003eTP53\u003c/em\u003e mutation, (NM_000546.6):c.713G\u0026gt;A, p.(Cys238Tyr), was also found in the mother\u0026rsquo;s CRC (Table 1). In the proband\u0026rsquo;s bilateral parotid tumours, copy-number neutral loss of heterozygosity (LOH) at the \u003cem\u003eAPC\u003c/em\u003e locus, extending to ~120Mb of chromosome 5q (Chr5q) was identified. However, the submandibular BCAC showed no Chr5q LOH (Figure 1 C-D; Figure S1) and instead carried a somatic \u003cem\u003eCTNNB1 \u003c/em\u003emutation,\u003cem\u003e \u003c/em\u003e(NM_001904.4):c.104T\u0026gt;C, p.(Ile35Thr) at 48.2%. Truncating \u003cem\u003eAPC\u003c/em\u003e mutations were also found in the proband\u0026rsquo;s and sister\u0026rsquo;s BCAs, but not in the proband\u0026rsquo;s IDH. Additionally, \u003cem\u003eBRAF\u003c/em\u003e mutation, (NM_004333.6):c.1799T\u0026gt;A, p.(Val600Glu) was detected in the proband\u0026rsquo;s PTC (Table 1). \u003c/p\u003e\n\n\u003cp\u003eTo further examine the relationship between the different salivary gland tumour subtypes and their expression profiles, spatial transcriptomic analyses were carried out on the proband\u0026rsquo;s lesions. Pan-CK+ AOIs in the IDH, BCA, parotid BCAC, submandibular BCAC and normal salivary gland tissue were selected for the analysis (Figure 2). UMAP analysis revealed two clusters, benign and malignant tissues, based on the expression of 16,489 genes (Figure S2). GSEA consistently identified the WNT signalling pathway among the top enriched gene-sets when comparing the tumour subtypes and normal salivary tissue (Figure S3). \u003c/p\u003e\n\n\u003cp\u003eSubsequently, an unsupervised hierarchical clustering analysis using the expression levels of the main WNT genes only (n=89) [14] yielded five distinct clusters, one per tumour/tissue subtype, with the two main branches separating the malignant and benign tissues. \u003cem\u003eCTNNB1\u003c/em\u003e expression levels were significantly different (t-test, p\u0026lt;0.05) between each subgroup, apart from the BCA and IDH, whereas \u003cem\u003eAPC\u003c/em\u003e expression levels were similar across all subtypes (Figure 3). WNT-GSEA scores were used to quantify the WNT activation associated to each tumour subtype (from IDH to BCA to BCACs) and their molecular background. There was an overall increase in WNT activation from IDH [no \u003cem\u003eCTNNB1 \u003c/em\u003ep.(Ile35Thr] or second hit in \u003cem\u003eAPC\u003c/em\u003e) to BCA [\u003cem\u003eAPC \u003c/em\u003ec.1161_1162del,\u003cem\u003e \u003c/em\u003ep.(His388Glnfs*8)] to parotid BCAC (Chr5q LOH) to submandibular BCAC [\u003cem\u003eCTNNB1\u003c/em\u003e p.(Ile35Thr)] (Figure S4). \u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eBCAs and BCACs are morphologically similar, with the only distinction being the invasive tumour growth beyond the capsule in BCACs [15]. Malignant transformation of BCA to BCAC has been proposed, analogous to the suggested role of IDH as a precursor to BCA [16,17]. The nuclear b-catenin staining seen in IDH, BCAs and BCACs indicates WNT pathway involvement. However, activating \u003cem\u003eCTNNB1\u003c/em\u003e mutations have primarily been observed in BCAs (50%; n=88/176) but only occasionally in BCACs (13%; n =4/30) and IDH (20%; n=3/15) (Tables S1-S2) [5,15,17-23]. Recently, \u003cem\u003eFBXW11\u003c/em\u003e, also a WNT family gene, was identified as a new driver in BCAs lacking \u003cem\u003eCTNNB1\u003c/em\u003e variants [23]. In a limited number of BCACs, variants in \u003cem\u003eCYLD\u003c/em\u003e, \u003cem\u003ePIK3CA\u003c/em\u003e and \u003cem\u003eHRAS\u003c/em\u003e have been reported, but not in a WNT gene other than \u003cem\u003eCTNNB1 \u003c/em\u003eor\u003cem\u003e FBXW11 \u003c/em\u003e[3,15,20,23]. \u003c/p\u003e\n\u003cp\u003eIn the familial case presented herein, a germline deletion in \u003cem\u003eAPC\u003c/em\u003e and somatic mutations in either \u003cem\u003eAPC\u003c/em\u003e or \u003cem\u003eCTNNB1\u003c/em\u003e were identified in the salivary and colon tumours, partially fitting the b-catenin/WNT signalling model (Table 1). The APC domain involved in b-catenin binding and degradation consists of seven (14 with both alleles) 20-amino-acid repeats (20-AARs) [24]. In FAP/AFAP tumours, \u003cem\u003eAPC\u003c/em\u003e PVs typically result in a truncated protein retaining one to three 20-AARs, encoded by codon 1250-1450 and known as the mutation cluster region. This ensures the \u0026lsquo;just-right\u0026rsquo; amount of \u0026beta;-catenin regulation is maintained, promoting an optimal level of WNT activity for tumorigenesis [24,25]. In the patients described, only the proband\u0026rsquo;s BCA and mother\u0026rsquo;s colon polyp followed this \u0026lsquo;just-right\u0026rsquo; model, by retaining three 20-AARs, whereas the other tumours retained four to ten 20-AARs (Figure 1C, Table S3). \u003c/p\u003e\n\u003cp\u003eHistopathological studies have indicated a possible progression from BCA to BCAC [16,26,27]. Although the numbers of cases are limited, the aberrations in genes affecting different signalling pathways in BCAC and BCA suggest that most BCACs likely arise \u003cem\u003ede novo\u003c/em\u003e and not from BCA, although there is some overlap and more studies are required [15,20,23]. In our proband, the absence of shared somatic variants among the salivary tumours and the distinct second hits detected in each lesion suggest that they developed independently and thus, the BCACs did not arise from the BCA (Figure 1C; Table 1). Rather, multiple primary tumours at different stages of tumorigenesis were observed, which is in-line with the predisposition to multiple cancers caused by a germline \u003cem\u003eAPC\u003c/em\u003e deletion. \u003c/p\u003e\n\u003cp\u003eSpatial transcriptomic analysis of the proband\u0026rsquo;s salivary gland lesions and normal tissue showed distinct clustering based on subtype, with WNT scores progressing from benign to malignant tissues (Figure 3; Figure S4). The increase in WNT scores reflects the gradient in aggressivity expected from benign lesion (IDH) to benign tumour (BCA) to malignant tumours (BCACs). Interestingly, the submandibular BCAC with the somatic \u003cem\u003eCTNNB1\u003c/em\u003e mutation, p.(Ile35Thr), had a higher WNT score than the parotid BCAC with Chr5q LOH. Remarkably, the \u003cem\u003eCTNNB1\u003c/em\u003e p.(Ile35Thr) mutation is almost exclusively seen in salivary gland tumours (94%; n=72/77) (c-square test, p\u0026lt;10\u003csup\u003e-15\u003c/sup\u003e) (Table S4-5) [5,15,17-23,28,29]. This suggests that the \u003cem\u003eCTNNB1\u003c/em\u003e p.(Ile35Thr) mutation somatically can lead to BCAC development and has a greater impact on WNT dysregulation than does \u003cem\u003eAPC\u003c/em\u003e inactivation, possibly due to its high specificity to salivary gland tumours. \u003c/p\u003e\n\u003cp\u003eIn summary, spatial transcriptomic analysis revealed a stepwise increase in WNT activation from IDH to BCA to BCAC. Additionally, the proband\u0026rsquo;s BCACs did not appear to arise from the BCA. Our findings showcase salivary gland tumours, BCA and BCAC, as new phenotypes of AFAP, highlighting that more than 30 years since the discovery of \u003cem\u003eAPC\u003c/em\u003e as the cause of FAP, new phenotypes are still being uncovered. \u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: \u003c/p\u003e\n\u003cp\u003eWe would like to thank Tamiko Nishimura, Naciba Benlimame and Lilian Canetti for their support in the spatial transcriptomic assay. We thank Anne-Sophie Chong for her help sequencing the PTC and Jes\u0026uacute;s del Valle for his assistance in evaluating the pathogenicity of the germline \u003cem\u003eAPC\u003c/em\u003e deletion. We acknowledge the molecular genetics laboratory team of the Institute of Human Genetics in Ulm for their help with the OncoScan and CytoScan assays.\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Canadian Institutes of Health Research grant (FDN-148390) to WDF. BR is a Miguel Servet Fellow (CP21/00038) from the Instituto de Salud Carlos III (PI20/01721). FCPC is supported by the George G Harris Fellowship. JCV was supported in part by an internal award from The Research Institute of the McGill University Centre (RI-MUHC).\u003c/p\u003e\n\u003cp\u003eThis research as well as biobanking of biological material and data was made possible in part through a collaboration with the R\u0026eacute;seau de recherche sur le cancer (RRCancer) financially supported by the Oncopole, the FRQ cancer division, which receives funding from Merck Canada Inc., GSK, Pfizer and the Minist\u0026egrave;re de l\u0026apos;\u0026Eacute;conomie, de l\u0026apos;Innovation et de l\u0026apos;\u0026Eacute;nergie du Qu\u0026eacute;bec. The RRCancer is affiliated to the Canadian Tumor Repository Network (CTRNet).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e: \u003c/p\u003e\n\u003cp\u003eFCPC collected the samples, carried out the genomic and spatial transcriptomic experiments and analyses and drafted the manuscript. CB, FT, JL and TCM provided the patient samples and clinical information. SD and RS conducted the OncoScan and CytoScan analyses. MP and JC performed pathology reviews and participated in the spatial transcriptomic assay. JCV and PP assisted with the genomic analyses. BR and WDF oversaw all aspects of the project and edited the manuscript. All authors approved the submission of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement\u003c/strong\u003e: \u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study will be deposited in will be provided in the final version of the manuscript and made publicly available prior to publication. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTo VSH, Chan JYW, Tsang RKY\u003cem\u003e, et al.\u003c/em\u003e Review of Salivary Gland Neoplasms. \u003cem\u003eInternational Scholarly Research Notices \u003c/em\u003e2012; \u003cstrong\u003e2012\u003c/strong\u003e: 872982.\u003c/li\u003e\n\u003cli\u003eAgulnik M, McGann CF, Mittal BB\u003cem\u003e, et al.\u003c/em\u003e Management of salivary gland malignancies: current and developing therapies. \u003cem\u003eOncology Reviews \u003c/em\u003e2008; \u003cstrong\u003e2\u003c/strong\u003e: 86-94.\u003c/li\u003e\n\u003cli\u003eRobinson RA. 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CTNNB1 mutations in basal cell adenoma of the salivary gland. \u003cem\u003eJ Formos Med Assoc \u003c/em\u003e2018; \u003cstrong\u003e117\u003c/strong\u003e: 894-901.\u003c/li\u003e\n\u003cli\u003eOyama Y, Nishida H, Kusaba T\u003cem\u003e, et al.\u003c/em\u003e Difference in transducin-like enhancer of split 1 protein expression between basal cell adenomas and basal cell adenocarcinomas - an immunohistochemical study. \u003cem\u003eDiagn Pathol \u003c/em\u003e2018; \u003cstrong\u003e13\u003c/strong\u003e: 48.\u003c/li\u003e\n\u003cli\u003eWong K, Bishop JA, Weinreb I\u003cem\u003e, et al.\u003c/em\u003e Activation of Wnt/\u0026beta;-catenin signalling by mutually exclusive FBXW11 and CTNNB1 hotspot mutations drives salivary gland basal cell adenoma. \u003cem\u003ebioRxiv \u003c/em\u003e2024: 2024.2010.2021.619442.\u003c/li\u003e\n\u003cli\u003eCrabtree M, Sieber OM, Lipton L\u003cem\u003e, et al.\u003c/em\u003e Refining the relation between \u0026lsquo;first hits\u0026rsquo; and \u0026lsquo;second hits\u0026rsquo; at the APC locus: the \u0026lsquo;loose fit\u0026rsquo; model and evidence for differences in somatic mutation spectra among patients. \u003cem\u003eOncogene \u003c/em\u003e2003; \u003cstrong\u003e22\u003c/strong\u003e: 4257-4265.\u003c/li\u003e\n\u003cli\u003eChristie M, Jorissen RN, Mouradov D\u003cem\u003e, et al.\u003c/em\u003e Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/\u0026beta;-catenin signalling thresholds for tumourigenesis. \u003cem\u003eOncogene \u003c/em\u003e2013; \u003cstrong\u003e32\u003c/strong\u003e: 4675-4682.\u003c/li\u003e\n\u003cli\u003eBatsakis JG, Luna MA. Basaloid Salivary Carcinoma. \u003cem\u003eAnnals of Otology, Rhinology \u0026amp; Laryngology \u003c/em\u003e1991; \u003cstrong\u003e100\u003c/strong\u003e: 785-787.\u003c/li\u003e\n\u003cli\u003eQiao C, Li D, Zhang Z\u003cem\u003e, et al.\u003c/em\u003e Intracapsular carcinoma ex basal cell adenoma of the parotid gland. \u003cem\u003eInternational Journal of Oral and Maxillofacial Surgery \u003c/em\u003e2020; \u003cstrong\u003e49\u003c/strong\u003e: 1548-1550.\u003c/li\u003e\n\u003cli\u003eDaa T, Kashima K, Kaku N\u003cem\u003e, et al.\u003c/em\u003e Mutations in components of the Wnt signaling pathway in adenoid cystic carcinoma. \u003cem\u003eMod Pathol \u003c/em\u003e2004; \u003cstrong\u003e17\u003c/strong\u003e: 1475-1482.\u003c/li\u003e\n\u003cli\u003eDaa T, Kaku N, Kashima K\u003cem\u003e, et al.\u003c/em\u003e Expression of beta-catenin, E-cadherin and cyclin D1 in adenoid cystic carcinoma of the salivary gland. \u003cem\u003eJ Exp Clin Cancer Res \u003c/em\u003e2005; \u003cstrong\u003e24\u003c/strong\u003e: 83-87.\u003c/li\u003e\n\u003cli\u003eCameselle-Teijeiro JM, Peteiro-Gonz\u0026aacute;lez D, Caneiro-G\u0026oacute;mez J\u003cem\u003e, et al.\u003c/em\u003e Cribriform-morular variant of thyroid carcinoma: a neoplasm with distinctive phenotype associated with the activation of the WNT/\u0026beta;-catenin pathway. \u003cem\u003eMod Pathol \u003c/em\u003e2018; \u003cstrong\u003e31\u003c/strong\u003e: 1168-1179.\u003c/li\u003e\n\u003cli\u003eNieminen TT, Walker CJ, Olkinuora A\u003cem\u003e, et al.\u003c/em\u003e Thyroid Carcinomas That Occur in Familial Adenomatous Polyposis Patients Recurrently Harbor Somatic Variants in APC, BRAF, and KTM2D. \u003cem\u003eThyroid \u003c/em\u003e2020; \u003cstrong\u003e30\u003c/strong\u003e: 380-388.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"McGill University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Familial adenomatous polyposis, APC, WNT pathway, Basal cell adenocarcinoma, Basal cell adenoma, spatial transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-6557806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6557806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAttenuated familial adenomatous polyposis (AFAP) is a disorder caused by germline pathogenic variants in \u003cem\u003eAPC\u003c/em\u003e and is characterized by the presence of \u0026lt;100 colonic polyps and a high lifetime risk of developing colorectal cancer. Salivary gland basal cell tumours are uncommon and have not been reported in AFAP before. We present a family with AFAP and multiple salivary gland tumours, including basal cell adenoma (BCA) and basal cell adenocarcinoma (BCAC). The colon and salivary gland tumours showed abnormal nuclear beta-catenin staining. Genomic analysis of both parotid BCACs showed CNN-LOH at the \u003cem\u003eAPC\u003c/em\u003e locus, implicating loss of full-length APC in the aetiology of the parotid BCACs. In contrast, the submandibular BCAC showed a p.(Ile35Thr) \u003cem\u003eCTNNB1\u003c/em\u003e mutation. Spatial transcriptomic analysis revealed a stepwise increase in the expression of WNT pathway genes across the proband’s salivary lesions, from benign (intercalated duct hyperplasia and BCA) to malignant (BCACs). Our results showcase BCA and BCAC as new phenotypes of AFAP.\u003c/p\u003e","manuscriptTitle":"APC-related multiple salivary gland lesions: spatial transcriptomic analysis reveals progressive WNT activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 11:34:28","doi":"10.21203/rs.3.rs-6557806/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b849279-7b1d-4558-9d19-cdd412f502c0","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47888995,"name":"Cancer Biology"},{"id":47888996,"name":"Molecular Genetics"},{"id":47888997,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2025-05-05T11:34:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 11:34:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6557806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6557806","identity":"rs-6557806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}