CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation

CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation Silvia González-Martínez, Viera Horvathova Kajabova, Belén Pérez-Mies, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3416058/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2024 Read the published version in Virchows Archiv → Version 1 posted You are reading this latest preprint version Abstract The proposed role of CDH1 (E-cadherin gene) methylation as a mechanism of gene inactivation in Invasive Lobular Carcinoma (ILC) remains inconclusive. Using pyrosequencing, we analyzed CDH1 hypermethylation in E-cadherin deficient ILC cases with varied CDH1 mutation status and Invasive Breast Carcinomas Non-Special Type (IBC-NSTs), finding no hypermethylation in any group of ILCs. In addition, CDH1 methylation correlated with the presence of Tumor-Infiltrating Lymphocytes (TILs). These findings challenge CDH1 methylation as a CDH1 inactivation mechanism in ILC and highlight TILs as a potential confounding factor in the gene methylation. Breast cancer lobular carcinomas CDH1 gene methylation Figures Figure 1 Figure 2 Introduction Invasive Lobular Carcinoma (ILC) is the second most common type of invasive breast cancer, accounting for around 10–15% of all cases. ILC is characterized by its unique growth pattern. The key molecular hallmark is the loss of the epithelial specific cell-cell adhesion molecule E-cadherin, encoded by CDH1 , which occurs in the 85% of ILC[ 1 ]. The molecular mechanisms involved in the decrease or even loss of this protein can be variable. The CDH1 gene can be inactivated by mutations (50–60% cases) and loss of heterozygosity[ 1 ]. For many years, CDH1 promoter hypermethylation has been accepted as a mechanism for gene inactivation in ILC. This assumption largely stems from non-quantitative assays, predominantly Methylation-Specific PCR (MSP), which reported CDH1 hypermethylation frequencies ranging from 26 to 93%. However, it has been demonstrated that MSP can yield a significant number of false-positive results[ 2 ]. The comprehensive TCGA study by Ciriello et al.[ 3 ] challenged the hypothesis of frequent CDH1 methylation in ILC. Analyzing 111 ILCs via Illumina Infinium DNA methylation HumanMethylation 27 (HM27) and HumanMethylation 450 (HM450) platforms, the authors unveiled similar CDH1 methylation patterns in ILCs and invasive breast carcinomas non-special type (IBC-NSTs), the latter characterized by preserved E-cadherin expression. While the study encompassed both CDH1 wild-type and mutated ILCs, it lacked a dedicated analysis of methylation data stratified by mutation status or CDH1 mRNA expression, positive in 13% of samples. More recently, Alexander et al.[ 4 ] also failed to identify significant CDH1 promoter methylation in 9 ILC cases exhibiting varying levels of E-cadherin expression through methylation EPIC BeadChip 850K array analysis. In light of these discoveries, we hypothesized that if CDH1 methylation contributes to CDH1 gene inactivation in ILCs, it would be more prevalent in tumors lacking E-cadherin expression and devoid of CDH1 mutation. In this selected group of cases, CDH1 methylation could presents itself as a viable alternative mechanism for inducing inactivation, complementing the role typically fulfilled by gene mutations. Pyrosequencing is a high-resolution method for the detection of DNA methylation and provides quantitative information for each CpG site under study, allowing for the control of bisulfite conversion efficiency. Pyrosequencing is the technique with the best reproducibility (even higher than methylation array) and can work well even on minute amounts of highly fragmented DNA[ 5 ]. Methods The study received approval from the Local Ethics Committee (Ramón y Cajal Research Ethics Committee reference 223/18). A total of 36 cases were selected from the Pathology Department of Ramón y Cajal University Hospital (Madrid, Spain). Clinical data were obtained from clinical databases. Histological evaluation, immunohistochemistry, and sequencing were carried out as previously reported[ 6 ]. Tumor-Infiltrating Lymphocytes (TILs) evaluation was conducted in regions where DNA was extracted for methylation analysis, following the recommendations of the TILs Working Group[ 7 ]. Genomic DNA (2 µg) from all tumors was used for sodium bisulfite treatment using the EpiTect Bisulfite kit (Qiagen). This approach ensures the complete conversion of unmethylated cytosine to uracil, enabling the detection of methylated CpGs. Four sets of primers were designed, covering 18 CpG dinucleotides in the regulatory regions of the gene CDH1 – N-shore, CpG Island, and S-shore, using PyroMark Assay Design 2.0 software (Qiagen) (Supplementary Table 1). Quantitative pyrosequencing was employed to assess the DNA methylation of these regulatory regions. PCR amplification was conducted with PyroMark PCR Kit (Qiagen) as per the manufacturer’s instructions. Pyrosequencing was performed using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 platform. Data analysis utilized the PyroMark Q24 2.0.6. software (Qiagen) and the methylation plotter web tool[ 8 ]. Median methylation values for each CpG were compared among the three groups of tumors (Kruskal-Wallis or ANOVA test). To examine differences in methylation levels across studied regions, the median of the mean methylation values of the CpG sites per region were compared among groups (Kruskal-Wallis or ANOVA test). Results and Discussion To ascertain the prevalence of CDH1 methylation in ILCs characterized by both the absence of CDH1 mutation in the exonic region and E-cadherin expression, we conducted quantitative pyrosequencing on a cohort of 17 ILC cases that had undergone comprehensive massive parallel sequencing, revealing a lack of CDH1 mutations and complete E-cadherin expression absence[ 6 , 9 , 10 ]. For comparative purposes, we analyzed 10 ILC cases with CDH1 mutations and full E-cadherin expression loss, along with 9 IBC-NSTs marked by preserved E-cadherin expression and no CDH1 mutation. The main clinicopathological and molecular data of the patients are presented in Supplementary Table 2. Within this cohort of 36 primary tumors, we comprehensively examined the hypermethylation status of 18 CpG dinucleotides situated in the CpG island (103 pb) of the CDH1 gene and in the Northern and Southern shore (N-shore, S-shore) regions. The CpG island region encompassed the majority of sites explored in prior MSP studies as well as 4 CpGs scrutinized by Ciriello et al.[ 3 ] and 5 CpGs by Alexander et al.[ 4 ] through methylation arrays (Supplementary Tables 3, 4, 5). Notably, the observed methylation values in the CpG island were generally modest (ranging between 3 to 18%) (Fig. 1 A). CpG methylation values did not statistically differ among the studied groups for any of the analyzed CpG sites, except for CpG sites at positions 68737141 and 68737296 located in the CpG island region. Interestingly, these CpGs exhibited slightly heightened methylation levels in IBC-NSTs (p-value < 0.05) (Supplementary Fig. 1). Furthermore, there were significant differences in methylation levels in the whole island region between the group of mutated ILCs and IBC-NSTs, the latter being higher (Fig. 1 B). Tissue- and cancer-specific differentially methylated regions can occur not only within CpG islands themselves but also within CpG island shores, regions of relatively low CpG density, situated proximal to conventional promoter CpGs (up to 2 kb distant). This suggests the potential involvement of shore methylation in tissue differentiation, epigenetic reprogramming, and cancer[ 11 ]. Intriguingly, the analysis of CDH1 shores methylation has not been analyzed in MSP studies (Supplementary Table 3). Therefore, we extended our primer design to CpGs located in both N-shore and S-shore (Supplementary Table 3). Methylation levels in these regions were, in general, higher than in the CpG island (ranging from 4 to 35% and 14 to 64%, respectively) (Fig. 1 A). There were no significant differences between the studied groups, either in terms of CpG site-specific comparisons (Fig. 1 A) or whole region assessments (Fig. 1 B). In an effort to corroborate our findings, we compare our results with those reported by Ciriello et al.[ 3 ] and Alexander et al.[ 4 ]. Unfortunately, the available datasets from Ciriello et al. lacks explicit specification of methylation beta values corresponding to the individual probes, offering a graphical overview instead. Since they did not make a differential analysis between the methylation status of CDH1 -mutated and non-mutated cases, we compared the methylation frequencies at each CpG site for both ILC groups combined (with and without CDH1 mutation). Conversely, the dataset provided by Alexander et al.[ 4 ] allowed us to compare methylation levels in ILCs according to CDH1 mutation status, although the small number of cases lacking CDH1 mutation ( n = 4) was a significant limitation of data reproducibility. In general, we observed similarity in methylation levels when compared to those outlined by Ciriello et al. [ 3 ], while we demonstrated lower methylation levels in contrast to those observed in the study by Alexander et al.[ 4 ] (Supplementary Tables 4 and 5). Furthermore, Fridrichova et al.[ 12 ] reported CDH1 methylation levels assessed by pyrosequencing across 7 identical CpGs situated within the CpG island among 24 ILC cases, 178 invasive ductal carcinoma, and 4 other breast cancer patients. Although the mutational status of ILC cases was not assessed in this study, consistent with our current results, there were no disparities in DNA methylation across these groups, and the average value in tumors and paired lymph node metastasis remained below 10.5%[ 12 ]. While we did not observe substantial differences in CDH1 methylation across diverse tumor subtypes, noteworthy instances of elevated methylation were noted in selected tumors, such as case 11. The relevant aspect to be considered is that CDH1 methylation can occur in TILs, thereby introducing a confounding element that can lead to false positive outcomes, particularly when using MSP[ 2 ]. To confirm this hypothesis, we conducted a correlation analysis between TILs and methylation levels across different CpGs, unveiling a modest yet statistically significant correlation between TILs and methylation levels across all examined regions (p-value < 0.05) (Fig. 2 and Supplementary Fig. 2). In conclusion, our findings, facilitated by high-resolution quantitative detection methodology, indicated that the frequency and extent of CDH1 gene methylation in ILCs is not higher than those observed in IBC-NSTs. This result held true irrespective of the presence or absence of CDH1 mutations, thereby challenging the notion of CDH1 methylation as a pervasive mechanism for CDH1 gene inactivation. Moreover, our analysis suggested the potential impact of TILs abundance on CDH1 methylation analysis. Importantly, the conspicuous loss of E-cadherin in the non-mutated ILC subgroup might be driven by mechanisms beyond DNA methylation. The intricate interplay of additional epigenetic mechanisms and non-genetic determinants, such as cellular signaling pathways, environmental factors, and cellular context, holds promise in shedding light on this phenomenon. Abbreviations Invasive Lobular Carcinoma (ILC), Invasive Breast Carcinomas Non-Special Type (IBC-NSTs), Tumor-Infiltrating Lymphocytes (TILs) , Methylation-Specific PCR (MSP) , HumanMethylation 27 (HM27) , HumanMethylation 450 (HM450) , Northern shore (N-shore) , Southern shore (S-shore). Declarations Author contributions: SG-M carried out most of the experimental process, the statistical analyses, the preparation of the original manuscript and contributed to the conceptualization stage. VHK contributed in the experimental part, including primers design. JP and BS acted as corresponding authors, were responsible for the conceptualization and contributed to the process of writing and revising the manuscript, and ensured the consistency of the manuscript. BP-M, ICB and JP performed the histological examination of the tumors. DS, GM-B, MG, JP-G and JC reviewed and provided critical input to the original manuscript. All authors read and approved the final manuscript. Ethical Approval The study received approval from the Local Ethics Committee (Ramón y Cajal Research Ethics Committee reference 223/18) at the Ramón y Cajal University Hospital, 28034 Madrid, Spain. Funding This study was funded by grants from the Instituto de Salud Carlos III (ISCIII) (PI19/01331 and PI22/01892) and was realized thanks to the Short-Term Scientific Mission Grant awarded by the Cost action CA19138 to SG-M. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Availability of data and materials Additional information on this article can be found in supplementary material. Competing interests: Consulting/Advisor: Roche , AstraZeneca, Seattle Genetics, Daiichi Sankyo, Lilly, Merck Sharp&Dohme, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Scorpion Therapeutics, Expres2ion Biotechnologies, Jazz Pharmatheuticals, Abbvie. Honoraria: Roche , Novartis , Eisai, Pfizer, Lilly, Merck Sharp&Dohme, Daiichi Sankyo, Astrazeneca, Gilead, Steamline Therapeutics. Research funding to the Institution: Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer healthcare, Eisai, F.Hoffman-La Roche, Guardanth health, Merck Sharp&Dohme, Pfizer, Piqur Therapeutics, Queen Mary University of London, IQVIA. Stock: MAJ3 Capital, Leuko (relative). Travel, accommodation, expenses: Roche, Novartis, Eisai, pfizer, Daiichi Sankyo, Astrazeneca, Gilead, Merck Sharp&Dohme, Steamline. Patents: Pharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent.Javier Cortés Castán, Alejandro Piris Giménez, Violeta Serra Elizalde. WO 2014/199294 A. ISSUED . Her2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy.Aleix Prat, Antonio Llombart, Javier Cortés.US 2019/ 0338368 A1. LICENSED References WHO Classification of Tumours Editorial Board, International Agency for Research on Cancer, World Health Organization. WHO classification of tumours. Breast Tumours. Lyon: International Agency for Research on Cancer; 2019. Bücker L, Lehmann U. CDH1 (E-cadherin) Gene Methylation in Human Breast Cancer: Critical Appraisal of a Long and Twisted Story. Cancers. 2022;14:4377. Ciriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al. Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer. Cell. 2015;163:506–19. Alexander J, Mariani O, Meaudre C, Fuhrmann L, Xiao H, Naidoo K, et al. Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer. Cancers. 2022;14:295. The BLUEPRINT consortium, Bock C, Halbritter F, Carmona FJ, Tierling S, Datlinger P, et al. Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nat Biotechnol. 2016;34:726–37. González-Martínez S, Pizarro D, Pérez-Mies B, Caniego-Casas T, Rodríguez-Peralto JL, Curigliano G, et al. Differences in the Molecular Profile between Primary Breast Carcinomas and Their Cutaneous Metastases. Cancers. 2022;14:1151. Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Annals of Oncology. 2015;26:259–71. Mallona I, Díez-Villanueva A, Peinado MA. Methylation plotter: a web tool for dynamic visualization of DNA methylation data. Source Code Biol Med. 2014;9:11. Rosa-Rosa J, Caniego-Casas T, Leskela S, Cristobal E, González-Martínez S, Moreno-Moreno E, et al. High Frequency of ERBB2 Activating Mutations in Invasive Lobular Breast Carcinoma with Pleomorphic Features. Cancers. 2019;11:74. Pérez-Mies B, Caniego-Casas T, Carretero-Barrio I, Biscuola M, López-García MA, Hardisson D, et al. The Clonal Relationship Between the Ductal and Lobular Components of Mixed Ductal-Lobular Carcinomas Suggested a Ductal Origin in Most Tumors. American Journal of Surgical Pathology. 2022;46:1545–53. Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet. 2009;41:178–86. Fridrichova I, Smolkova B, Kajabova V, Zmetakova I, Krivulcik T, Mego M, et al. CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers. Translational Research. 2015;165:717–30. Additional Declarations Competing interest reported. Competing interests (JC): Consulting/Advisor: Roche , AstraZeneca, Seattle Genetics, Daiichi Sankyo, Lilly, Merck Sharp&Dohme, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Scorpion Therapeutics, Expres2ion Biotechnologies, Jazz Pharmatheuticals, Abbvie. Honoraria: Roche , Novartis , Eisai, Pfizer, Lilly, Merck Sharp&Dohme, Daiichi Sankyo, Astrazeneca, Gilead, Steamline Therapeutics. Research funding to the Institution: Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer healthcare, Eisai, F.Hoffman-La Roche, Guardanth health, Merck Sharp&Dohme, Pfizer, Piqur Therapeutics, Queen Mary University of London, IQVIA. Stock: MAJ3 Capital, Leuko (relative). Travel, accommodation, expenses: Roche, Novartis, Eisai, pfizer, Daiichi Sankyo, Astrazeneca, Gilead, Merck Sharp&Dohme, Steamline. Patents: Pharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent.Javier Cortés Castán, Alejandro Piris Giménez, Violeta Serra Elizalde. WO 2014/199294 A. ISSUED. Her2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy.Aleix Prat, Antonio Llombart, Javier Cortés.US 2019/ 0338368 A1. LICENSED Supplementary Files SupplementalmaterialSGMCE.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 May, 2024 Read the published version in Virchows Archiv → 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies 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-3416058","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":239433936,"identity":"d704d690-1ec7-42ee-9e8f-7149cd74a6ad","order_by":0,"name":"Silvia González-Martínez","email":"","orcid":"","institution":"Ramón y Cajal Health Research Institute (IRYCIS)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"González-Martínez","suffix":""},{"id":239433937,"identity":"f494ef15-5e31-4f92-971c-2c7dc2aae5c1","order_by":1,"name":"Viera Horvathova Kajabova","email":"","orcid":"","institution":"Biomedical Research Center of the Slovak Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Viera","middleName":"Horvathova","lastName":"Kajabova","suffix":""},{"id":239433938,"identity":"7c54fce5-8462-4163-9aba-fba97b9b97c6","order_by":2,"name":"Belén Pérez-Mies","email":"","orcid":"","institution":"Ramón y Cajal University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Belén","middleName":"","lastName":"Pérez-Mies","suffix":""},{"id":239433940,"identity":"d890c528-b1d8-4a95-ac45-a15c44f60d64","order_by":3,"name":"Irene Carretero-Barrio","email":"","orcid":"","institution":"Ramón y Cajal Health Research Institute (IRYCIS)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Carretero-Barrio","suffix":""},{"id":239433942,"identity":"3e32741a-c9aa-4b0b-a4a0-d6acfbd274a3","order_by":4,"name":"David Sarrió","email":"","orcid":"","institution":"Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas 'Alberto Sols', Conexión Cáncer (UAM-CSIC)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Sarrió","suffix":""},{"id":239433943,"identity":"9c98fa5e-0ceb-46b4-8b3a-3d392c612193","order_by":5,"name":"Gema Moreno-Bueno","email":"","orcid":"","institution":"Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas 'Alberto Sols', Conexión Cáncer (UAM-CSIC)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gema","middleName":"","lastName":"Moreno-Bueno","suffix":""},{"id":239433944,"identity":"3419aebf-a6fc-4e3a-878f-5b85e24d4720","order_by":6,"name":"María Gión","email":"","orcid":"","institution":"Ramón y Cajal University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Gión","suffix":""},{"id":239433946,"identity":"dbf2659d-dce8-48bc-aa31-f05fd3159892","order_by":7,"name":"José Perez-García","email":"","orcid":"","institution":"International Breast Cancer Center (IBCC)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Perez-García","suffix":""},{"id":239433948,"identity":"2cd11129-9e6c-4b45-ae42-5166d7b0c5fc","order_by":8,"name":"Javier Cortés","email":"","orcid":"","institution":"International Breast Cancer Center (IBCC)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Cortés","suffix":""},{"id":239433949,"identity":"a07ce23e-ce7a-4b55-ac3f-dff05cfb97ea","order_by":9,"name":"Bozena Smolkova","email":"","orcid":"","institution":"Biomedical Research Center of the Slovak Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bozena","middleName":"","lastName":"Smolkova","suffix":""},{"id":239433950,"identity":"6b0c4992-075d-4cb0-b8ed-0fd0579e53e1","order_by":10,"name":"José Palacios","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABN0lEQVRIiWNgGAWjYBACxgYILQNhVjAw8DEwGAAFmBnYGBLwauGBMM8wAFUS0AIDPBAD2pC0MODQwtze/vAB4w47Hv72w40Pf86rk2eTSN7AdKPGOp+PPYHxccUvTIf1nDE2YDyTzCNxJrHZmHfbYcM2ibQC5pxj6ZZtPA+YDc/2YWqZkcMmwdjGzMNwg7FNmnHbAcY2iRwD5hy2wwZsEglsko09WLSkP//B2FbPIw/UIvlzTp09RMs/fFoSzIC+PsxjANQiwdvAnAjWktsG1dLwA6tfJBLPHOcxBPmF59jh5DaeZwWHc/vSDdh4HjYbNjZgaDEEhtiHjzuq5eSOH3/48EdNnW0/e/LGxznfrA3k25MPPmz4g6kFZEoiulEHoE5oAMcUGpCHSuEEmLaMglEwCkbBiAMA/o5o/FgtETkAAAAASUVORK5CYII=","orcid":"","institution":"Ramón y Cajal Health Research Institute (IRYCIS)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Palacios","suffix":""}],"badges":[],"createdAt":"2023-10-06 11:29:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3416058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3416058/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00428-024-03814-8","type":"published","date":"2024-05-07T04:01:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44708167,"identity":"c9ec6b8d-1305-4e8d-ac51-092d43379433","added_by":"auto","created_at":"2023-10-16 16:58:36","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610778,"visible":true,"origin":"","legend":"\u003cp\u003ea) DNA methylation status of sequenced CpGs sites aligning with the \u003cem\u003eCDH1\u003c/em\u003egene.b) Violin plots depicting methylation data across each tumor subgroup within the respective region.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3416058/v1/93e75c837a9056612bc202d5.jpeg"},{"id":44708975,"identity":"f083f3e7-e3a2-45b6-8467-dc9533f6d277","added_by":"auto","created_at":"2023-10-16 17:06:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":675867,"visible":true,"origin":"","legend":"\u003cp\u003ea) Correlation between \u003cem\u003eCDH1\u003c/em\u003e methylation frequency and percentage of TILs across island region. b) Hematoxylin-eosin staining displaying the percentage of TILs (40%) in non-mutated ILC case number 11.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3416058/v1/2c2d37a1888076b2e0704988.jpeg"},{"id":56140976,"identity":"ac63b4a2-fb02-49a3-8525-52e63b847578","added_by":"auto","created_at":"2024-05-09 04:42:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":506596,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3416058/v1/725a1b17-ad6f-4f45-b010-78803fe65e33.pdf"},{"id":44708165,"identity":"7f51aa0c-617f-4246-b71d-7bcd2843a390","added_by":"auto","created_at":"2023-10-16 16:58:36","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":629861,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalmaterialSGMCE.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3416058/v1/dcba4539bc990bc77c76947d.xlsx"}],"financialInterests":"Competing interest reported. Competing interests (JC): Consulting/Advisor: Roche , AstraZeneca, Seattle Genetics, Daiichi Sankyo, Lilly, Merck Sharp\u0026Dohme, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Scorpion Therapeutics, Expres2ion Biotechnologies, Jazz Pharmatheuticals, Abbvie. Honoraria: Roche , Novartis , Eisai, Pfizer, Lilly, Merck Sharp\u0026Dohme, Daiichi Sankyo, Astrazeneca, Gilead, Steamline Therapeutics. Research funding to the Institution: Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer healthcare, Eisai, F.Hoffman-La Roche, Guardanth health, Merck Sharp\u0026Dohme, Pfizer, Piqur Therapeutics, Queen Mary University of London, IQVIA. Stock: MAJ3 Capital, Leuko (relative). Travel, accommodation, expenses: Roche, Novartis, Eisai, pfizer, Daiichi Sankyo, Astrazeneca, Gilead, Merck Sharp\u0026Dohme, Steamline. Patents: Pharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent.Javier Cortés Castán, Alejandro Piris Giménez, Violeta Serra Elizalde. WO 2014/199294 A. ISSUED. Her2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy.Aleix Prat, Antonio Llombart, Javier Cortés.US 2019/ 0338368 A1. LICENSED","formattedTitle":"CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInvasive Lobular Carcinoma (ILC) is the second most common type of invasive breast cancer, accounting for around 10\u0026ndash;15% of all cases. ILC is characterized by its unique growth pattern. The key molecular hallmark is the loss of the epithelial specific cell-cell adhesion molecule E-cadherin, encoded by \u003cem\u003eCDH1\u003c/em\u003e, which occurs in the 85% of ILC[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The molecular mechanisms involved in the decrease or even loss of this protein can be variable. The \u003cem\u003eCDH1\u003c/em\u003e gene can be inactivated by mutations (50\u0026ndash;60% cases) and loss of heterozygosity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor many years, \u003cem\u003eCDH1\u003c/em\u003e promoter hypermethylation has been accepted as a mechanism for gene inactivation in ILC. This assumption largely stems from non-quantitative assays, predominantly Methylation-Specific PCR (MSP), which reported \u003cem\u003eCDH1\u003c/em\u003e hypermethylation frequencies ranging from 26 to 93%. However, it has been demonstrated that MSP can yield a significant number of false-positive results[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe comprehensive TCGA study by Ciriello et al.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] challenged the hypothesis of frequent \u003cem\u003eCDH1\u003c/em\u003e methylation in ILC. Analyzing 111 ILCs via Illumina Infinium DNA methylation HumanMethylation 27 (HM27) and HumanMethylation 450 (HM450) platforms, the authors unveiled similar \u003cem\u003eCDH1\u003c/em\u003e methylation patterns in ILCs and invasive breast carcinomas non-special type (IBC-NSTs), the latter characterized by preserved E-cadherin expression. While the study encompassed both \u003cem\u003eCDH1\u003c/em\u003e wild-type and mutated ILCs, it lacked a dedicated analysis of methylation data stratified by mutation status or \u003cem\u003eCDH1\u003c/em\u003e mRNA expression, positive in 13% of samples. More recently, Alexander et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] also failed to identify significant \u003cem\u003eCDH1\u003c/em\u003e promoter methylation in 9 ILC cases exhibiting varying levels of E-cadherin expression through methylation EPIC BeadChip 850K array analysis. In light of these discoveries, we hypothesized that if \u003cem\u003eCDH1\u003c/em\u003e methylation contributes to \u003cem\u003eCDH1\u003c/em\u003e gene inactivation in ILCs, it would be more prevalent in tumors lacking E-cadherin expression and devoid of \u003cem\u003eCDH1\u003c/em\u003e mutation. In this selected group of cases, \u003cem\u003eCDH1\u003c/em\u003e methylation could presents itself as a viable alternative mechanism for inducing inactivation, complementing the role typically fulfilled by gene mutations.\u003c/p\u003e \u003cp\u003ePyrosequencing is a high-resolution method for the detection of DNA methylation and provides quantitative information for each CpG site under study, allowing for the control of bisulfite conversion efficiency. Pyrosequencing is the technique with the best reproducibility (even higher than methylation array) and can work well even on minute amounts of highly fragmented DNA[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe study received approval from the Local Ethics Committee (Ram\u0026oacute;n y Cajal Research Ethics Committee reference 223/18). A total of 36 cases were selected from the Pathology Department of Ram\u0026oacute;n y Cajal University Hospital (Madrid, Spain). Clinical data were obtained from clinical databases. Histological evaluation, immunohistochemistry, and sequencing were carried out as previously reported[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor-Infiltrating Lymphocytes (TILs) evaluation was conducted in regions where DNA was extracted for methylation analysis, following the recommendations of the TILs Working Group[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenomic DNA (2 \u0026micro;g) from all tumors was used for sodium bisulfite treatment using the EpiTect Bisulfite kit (Qiagen). This approach ensures the complete conversion of unmethylated cytosine to uracil, enabling the detection of methylated CpGs. Four sets of primers were designed, covering 18 CpG dinucleotides in the regulatory regions of the gene \u003cem\u003eCDH1\u003c/em\u003e \u0026ndash; N-shore, CpG Island, and S-shore, using PyroMark Assay Design 2.0 software (Qiagen) (Supplementary Table\u0026nbsp;1). Quantitative pyrosequencing was employed to assess the DNA methylation of these regulatory regions. PCR amplification was conducted with PyroMark PCR Kit (Qiagen) as per the manufacturer\u0026rsquo;s instructions. Pyrosequencing was performed using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 platform. Data analysis utilized the PyroMark Q24 2.0.6. software (Qiagen) and the methylation plotter web tool[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Median methylation values for each CpG were compared among the three groups of tumors (Kruskal-Wallis or ANOVA test). To examine differences in methylation levels across studied regions, the median of the mean methylation values of the CpG sites per region were compared among groups (Kruskal-Wallis or ANOVA test).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eTo ascertain the prevalence of \u003cem\u003eCDH1\u003c/em\u003e methylation in ILCs characterized by both the absence of \u003cem\u003eCDH1\u003c/em\u003e mutation in the exonic region and E-cadherin expression, we conducted quantitative pyrosequencing on a cohort of 17 ILC cases that had undergone comprehensive massive parallel sequencing, revealing a lack of \u003cem\u003eCDH1\u003c/em\u003e mutations and complete E-cadherin expression absence[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For comparative purposes, we analyzed 10 ILC cases with \u003cem\u003eCDH1\u003c/em\u003e mutations and full E-cadherin expression loss, along with 9 IBC-NSTs marked by preserved E-cadherin expression and no \u003cem\u003eCDH1\u003c/em\u003e mutation. The main clinicopathological and molecular data of the patients are presented in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eWithin this cohort of 36 primary tumors, we comprehensively examined the hypermethylation status of 18 CpG dinucleotides situated in the CpG island (103 pb) of the \u003cem\u003eCDH1\u003c/em\u003e gene and in the Northern and Southern shore (N-shore, S-shore) regions. The CpG island region encompassed the majority of sites explored in prior MSP studies as well as 4 CpGs scrutinized by Ciriello et al.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and 5 CpGs by Alexander et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] through methylation arrays (Supplementary Tables\u0026nbsp;3, 4, 5). Notably, the observed methylation values in the CpG island were generally modest (ranging between 3 to 18%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CpG methylation values did not statistically differ among the studied groups for any of the analyzed CpG sites, except for CpG sites at positions 68737141 and 68737296 located in the CpG island region. Interestingly, these CpGs exhibited slightly heightened methylation levels in IBC-NSTs (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Fig.\u0026nbsp;1). Furthermore, there were significant differences in methylation levels in the whole island region between the group of mutated ILCs and IBC-NSTs, the latter being higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTissue- and cancer-specific differentially methylated regions can occur not only within CpG islands themselves but also within CpG island shores, regions of relatively low CpG density, situated proximal to conventional promoter CpGs (up to 2 kb distant). This suggests the potential involvement of shore methylation in tissue differentiation, epigenetic reprogramming, and cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Intriguingly, the analysis of \u003cem\u003eCDH1\u003c/em\u003e shores methylation has not been analyzed in MSP studies (Supplementary Table\u0026nbsp;3). Therefore, we extended our primer design to CpGs located in both N-shore and S-shore (Supplementary Table\u0026nbsp;3). Methylation levels in these regions were, in general, higher than in the CpG island (ranging from 4 to 35% and 14 to 64%, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). There were no significant differences between the studied groups, either in terms of CpG site-specific comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) or whole region assessments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn an effort to corroborate our findings, we compare our results with those reported by Ciriello et al.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and Alexander et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Unfortunately, the available datasets from Ciriello et al. lacks explicit specification of methylation beta values corresponding to the individual probes, offering a graphical overview instead. Since they did not make a differential analysis between the methylation status of \u003cem\u003eCDH1\u003c/em\u003e-mutated and non-mutated cases, we compared the methylation frequencies at each CpG site for both ILC groups combined (with and without \u003cem\u003eCDH1\u003c/em\u003e mutation). Conversely, the dataset provided by Alexander et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] allowed us to compare methylation levels in ILCs according to \u003cem\u003eCDH1\u003c/em\u003e mutation status, although the small number of cases lacking \u003cem\u003eCDH1\u003c/em\u003e mutation (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4) was a significant limitation of data reproducibility. In general, we observed similarity in methylation levels when compared to those outlined by Ciriello et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], while we demonstrated lower methylation levels in contrast to those observed in the study by Alexander et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] (Supplementary Tables\u0026nbsp;4 and 5). Furthermore, Fridrichova et al.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] reported \u003cem\u003eCDH1\u003c/em\u003e methylation levels assessed by pyrosequencing across 7 identical CpGs situated within the CpG island among 24 ILC cases, 178 invasive ductal carcinoma, and 4 other breast cancer patients. Although the mutational status of ILC cases was not assessed in this study, consistent with our current results, there were no disparities in DNA methylation across these groups, and the average value in tumors and paired lymph node metastasis remained below 10.5%[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile we did not observe substantial differences in \u003cem\u003eCDH1\u003c/em\u003e methylation across diverse tumor subtypes, noteworthy instances of elevated methylation were noted in selected tumors, such as case 11. The relevant aspect to be considered is that \u003cem\u003eCDH1\u003c/em\u003e methylation can occur in TILs, thereby introducing a confounding element that can lead to false positive outcomes, particularly when using MSP[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To confirm this hypothesis, we conducted a correlation analysis between TILs and methylation levels across different CpGs, unveiling a modest yet statistically significant correlation between TILs and methylation levels across all examined regions (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, our findings, facilitated by high-resolution quantitative detection methodology, indicated that the frequency and extent of \u003cem\u003eCDH1\u003c/em\u003e gene methylation in ILCs is not higher than those observed in IBC-NSTs. This result held true irrespective of the presence or absence of \u003cem\u003eCDH1\u003c/em\u003e mutations, thereby challenging the notion of \u003cem\u003eCDH1\u003c/em\u003e methylation as a pervasive mechanism for \u003cem\u003eCDH1\u003c/em\u003e gene inactivation. Moreover, our analysis suggested the potential impact of TILs abundance on \u003cem\u003eCDH1\u003c/em\u003e methylation analysis. Importantly, the conspicuous loss of E-cadherin in the non-mutated ILC subgroup might be driven by mechanisms beyond DNA methylation. The intricate interplay of additional epigenetic mechanisms and non-genetic determinants, such as cellular signaling pathways, environmental factors, and cellular context, holds promise in shedding light on this phenomenon.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eInvasive Lobular Carcinoma (ILC), Invasive Breast Carcinomas Non-Special Type (IBC-NSTs), Tumor-Infiltrating Lymphocytes (TILs)\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eMethylation-Specific PCR (MSP)\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eHumanMethylation 27 (HM27)\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eHumanMethylation 450 (HM450)\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eNorthern shore (N-shore)\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eSouthern shore (S-shore).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eSG-M carried out most of the experimental process, the statistical analyses, the preparation of the original manuscript and contributed to the conceptualization stage. VHK contributed in the experimental part, including primers design. JP and BS acted as corresponding authors, were responsible for the conceptualization and contributed to the process of writing and revising the manuscript, and ensured the consistency of the manuscript. BP-M, ICB and JP performed the histological examination of the tumors. DS, GM-B, MG, JP-G and JC reviewed and provided critical input to the original manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study received approval from the Local Ethics Committee (Ram\u0026oacute;n y Cajal Research Ethics Committee reference 223/18) at the Ramón y Cajal University Hospital, 28034 Madrid, Spain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by grants from the Instituto de Salud Carlos III (ISCIII) (PI19/01331 and PI22/01892) and was realized thanks to the Short-Term Scientific Mission Grant awarded by the Cost action CA19138 to SG-M. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional information on this article can be found in supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConsulting/Advisor: Roche , AstraZeneca, Seattle Genetics, Daiichi Sankyo, Lilly, Merck Sharp\u0026amp;Dohme, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Scorpion Therapeutics, Expres2ion Biotechnologies, Jazz Pharmatheuticals, Abbvie. Honoraria: Roche , Novartis , Eisai, Pfizer, Lilly, Merck Sharp\u0026amp;Dohme, Daiichi Sankyo, Astrazeneca, Gilead, Steamline Therapeutics. Research funding to the Institution: Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer healthcare, Eisai, F.Hoffman-La Roche, Guardanth health, Merck Sharp\u0026amp;Dohme, Pfizer, Piqur Therapeutics, Queen Mary University of London, IQVIA. Stock: MAJ3 Capital, Leuko (relative). Travel, accommodation, expenses: Roche, Novartis, Eisai, pfizer, Daiichi Sankyo, Astrazeneca, Gilead, Merck Sharp\u0026amp;Dohme, Steamline. Patents: \u003cem\u003ePharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent.Javier Cort\u0026eacute;s Cast\u0026aacute;n, Alejandro Piris Gim\u0026eacute;nez, Violeta Serra Elizalde. WO 2014/199294 A. \u0026nbsp;ISSUED\u003c/em\u003e. \u003cem\u003eHer2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy.Aleix Prat, Antonio Llombart, Javier Cort\u0026eacute;s.US 2019/ 0338368 A1. LICENSED\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO Classification of Tumours Editorial Board, International Agency for Research on Cancer, World Health Organization. WHO classification of tumours. Breast Tumours. Lyon: International Agency for Research on Cancer; 2019. \u003c/li\u003e\n\u003cli\u003eB\u0026uuml;cker L, Lehmann U. CDH1 (E-cadherin) Gene Methylation in Human Breast Cancer: Critical Appraisal of a Long and Twisted Story. Cancers. 2022;14:4377. \u003c/li\u003e\n\u003cli\u003eCiriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al. Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer. Cell. 2015;163:506\u0026ndash;19. \u003c/li\u003e\n\u003cli\u003eAlexander J, Mariani O, Meaudre C, Fuhrmann L, Xiao H, Naidoo K, et al. Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer. Cancers. 2022;14:295. \u003c/li\u003e\n\u003cli\u003eThe BLUEPRINT consortium, Bock C, Halbritter F, Carmona FJ, Tierling S, Datlinger P, et al. Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nat Biotechnol. 2016;34:726\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Mart\u0026iacute;nez S, Pizarro D, P\u0026eacute;rez-Mies B, Caniego-Casas T, Rodr\u0026iacute;guez-Peralto JL, Curigliano G, et al. Differences in the Molecular Profile between Primary Breast Carcinomas and Their Cutaneous Metastases. Cancers. 2022;14:1151. \u003c/li\u003e\n\u003cli\u003eSalgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Annals of Oncology. 2015;26:259\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eMallona I, D\u0026iacute;ez-Villanueva A, Peinado MA. Methylation plotter: a web tool for dynamic visualization of DNA methylation data. Source Code Biol Med. 2014;9:11. \u003c/li\u003e\n\u003cli\u003eRosa-Rosa J, Caniego-Casas T, Leskela S, Cristobal E, Gonz\u0026aacute;lez-Mart\u0026iacute;nez S, Moreno-Moreno E, et al. High Frequency of ERBB2 Activating Mutations in Invasive Lobular Breast Carcinoma with Pleomorphic Features. Cancers. 2019;11:74. \u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-Mies B, Caniego-Casas T, Carretero-Barrio I, Biscuola M, L\u0026oacute;pez-Garc\u0026iacute;a MA, Hardisson D, et al. The Clonal Relationship Between the Ductal and Lobular Components of Mixed Ductal-Lobular Carcinomas Suggested a Ductal Origin in Most Tumors. American Journal of Surgical Pathology. 2022;46:1545\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eIrizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet. 2009;41:178\u0026ndash;86. \u003c/li\u003e\n\u003cli\u003eFridrichova I, Smolkova B, Kajabova V, Zmetakova I, Krivulcik T, Mego M, et al. CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers. Translational Research. 2015;165:717\u0026ndash;30. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Breast cancer, lobular carcinomas, CDH1 gene, methylation","lastPublishedDoi":"10.21203/rs.3.rs-3416058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3416058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe proposed role of \u003cem\u003eCDH1\u003c/em\u003e (E-cadherin gene) methylation as a mechanism of gene inactivation in Invasive Lobular Carcinoma (ILC) remains inconclusive. Using pyrosequencing, we analyzed \u003cem\u003eCDH1\u003c/em\u003e hypermethylation in E-cadherin deficient ILC cases with varied \u003cem\u003eCDH1\u003c/em\u003emutation status and Invasive Breast Carcinomas Non-Special Type (IBC-NSTs), finding no hypermethylation in any group of ILCs. In addition, \u003cem\u003eCDH1\u003c/em\u003e methylation correlated with the presence of Tumor-Infiltrating Lymphocytes (TILs). These findings challenge \u003cem\u003eCDH1\u003c/em\u003e methylation as a \u003cem\u003eCDH1\u003c/em\u003e inactivation mechanism in ILC and highlight TILs as a potential confounding factor in the gene methylation.\u003c/p\u003e","manuscriptTitle":"CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-16 16:58:31","doi":"10.21203/rs.3.rs-3416058/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":"5f9e9965-efc2-4965-8dab-c2ef4a29332b","owner":[],"postedDate":"October 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T04:01:00+00:00","versionOfRecord":{"articleIdentity":"rs-3416058","link":"https://doi.org/10.1007/s00428-024-03814-8","journal":{"identity":"virchows-archiv","isVorOnly":true,"title":"Virchows Archiv"},"publishedOn":"2024-05-07 04:01:00","publishedOnDateReadable":"May 7th, 2024"},"versionCreatedAt":"2023-10-16 16:58:31","video":"","vorDoi":"10.1007/s00428-024-03814-8","vorDoiUrl":"https://doi.org/10.1007/s00428-024-03814-8","workflowStages":[]},"version":"v1","identity":"rs-3416058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3416058","identity":"rs-3416058","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}