Mammary tumors are among the most prevalent and clinically significant neoplastic diseases in dogs, yet their molecular heterogeneity is poorly understood. This study aimed to characterize gene expression in synchronous tumors from the same dog and assess whether these tumors share molecular profiles. A transcriptomic study was conducted using RNA sequencing data from 179 canine mammary tumors to define transcriptomic groups through unsupervised consensus clustering and modules from gene co-expression network analysis. A subset of 45 dogs with synchronous tumors (n = 90) was then used to evaluate intra-individual transcriptomic concordance by comparing cluster assignment and module expression between tumor pairs, classified as malignant-malignant, benign-benign, and malignant-benign. Additionally, interclass correlation coefficients were applied to identify highly correlated genes within each category. The analysis identified five transcriptomic clusters based on overall gene-expression patterns. GSEA revealed that all five clusters potentially resemble molecular subtypes described in human breast cancer, including luminal-, basal-, and claudin low-like. We found that synchronous tumors were heterogeneous with respect to both cluster assignments and gene-expression patterns, indicating low intra-individual similarity. This heterogeneity was further supported by low interclass correlations across most genes in synchronous tumors, independent of tumor malignancy. Our findings demonstrate substantial molecular heterogeneity of canine mammary tumors, both between and within individuals. The results highlight the value of expression-based approaches for characterizing biologically relevant processes in mammary tumors.

Intratumor heterogeneity presents a major challenge in precision oncology for endometrial cancer (EC). Circulating tumor DNA (ctDNA) offers a minimally invasive method to monitor tumor evolution and therapeutic resistance. In this retrospective study, we evaluated a tumor-agnostic NGS panel to detect and track ctDNA in 18 EC patients and directly compared its performance with a tumor-informed ddPCR approach. ctDNA was detected by NGS in over 60% of plasma samples, while ddPCR showed higher positivity rates in paired samples (71.9% vs 62.5%), with overall concordance of 65.7% and fair agreement (Cohen's kappa = 0.23). The cfDNA-NGS panel identified a broad spectrum of alterations, including relapse-specific mutations indicative of clonal evolution, but showed lower sensitivity for low-frequency variants compared with ddPCR. Discordant cases, including false-negative results in both approaches, highlight the impact of assay sensitivity, target selection, and biological factors on ctDNA detection. ctDNA dynamics correlated with disease progression and treatment response, although detection was limited in cases with brain metastases. These findings support the utility of tumor-agnostic ctDNA monitoring in advanced EC and highlight the importance of assay quality and careful interpretation to address limitations such as clonal hematopoiesis and technical sensitivity.

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Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer, and up to 50% of the lesions remain non-invasive even if untreated. Both tumor and microenvironmental features are associated with disease progression, but the cellular interactions are poorly understood. We investigated DCIS of the clinical breast cancer subtypes; Triple Negative (TN) and Luminal A (LumA)-like, presumed to represent lesions with the most contrasting invasive potential, using spatial transcriptomic and multiplex immunofluorescence analyses. In the intraductal cancer cells, we found significant differences between the two subtypes in expression of genes related to energy metabolism and secreted proteins. In the periductal space, we found differences in the absolute density of CD4+ T cells, CD8+ T cells, and B cells between TN and LumA-like DCIS. Spatial cellular neighborhood analyses revealed an inverse relationship between CD4+ T cells and B cells. These data suggest specific biological properties of TN and LumA-like DCIS that might underpin differences in progression potential.

Rank signaling regulates mammary gland development and epithelial differentiation. While Rank is expressed in both basal and luminal cells, its basal-specific role is unclear. Here, using inducible basal-specific Rank expression and lineage tracing, we show that Rank signaling regulates basal cell identity in postnatal mammary glands. Increased basal Rank activity disrupts basal and luminal identities, causing aberrant luminal-like differentiation, lactation defects, and premalignant lesions composed of hybrid basal-derived cells that progress to basal and luminal adenocarcinomas. Conversely, Rank loss reduces tumor formation and also impairs cell identity. Mechanistically, proteomic, transcriptomic, and chromatin analyses reveal that Rank activation drives epigenetic remodeling, leading to basal identity loss and tumor initiation. A basal Rank gene signature correlates with ductal carcinoma in situ recurrence, as well as poor outcomes in luminal breast cancers. Thus, basal Rank-driven lineage infidelity promotes pre-invasive lesions and transition to invasive breast cancer in females.

Fibroblast growth factor receptor 1 (FGFR1) amplification is frequently observed in ER⁺/HER2⁻ breast cancer and has been linked to poor response to endocrine therapy. While FGFR1 has been implicated in therapeutic resistance, its role in regulating interferon (IFN) response in the context of endocrine resistance remains unclear. We investigated whether FGFR1 modulates the IFN response through the GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in tamoxifen-resistant breast cancer. We employed tamoxifen-resistant ER⁺ breast cancer cell lines and a tamoxifen-sensitive FGFR1-amplified patient-derived xenograft (PDX) model to examine the impact of FGFR1 inhibition on the IFN response through RNA sequencing, FGFR1 knockdown or pharmacologic inhibition, and functional assays. Clinical relevance was evaluated in breast cancer cohorts. Tamoxifen-resistant cells exhibited increased cytosolic, cGAS-positive dsDNA foci alongside elevated p-IRF3, indicating constitutive engagement of the cGAS-STING pathway. FGFR1 inhibition re-sensitized resistant cells to tamoxifen and induced IFN response gene expression, which was further amplified by tamoxifen co-treatment. FGFR1 inhibition similarly potentiated tamoxifen-induced IFN response gene expression in vivo in a FGFR1-amplified PDX model. Mechanistically, FGFR1 knockdown enhanced STING-mediated IFNB1 expression in response to cytosolic DNA. In the METABRIC breast cancer cohort, FGFR1 amplification was associated with poor prognosis specifically in tumors with high STING1 expression. These findings identify FGFR1 as a suppressor of cGAS-STING-mediated interferon response, driven by tamoxifen in ER⁺ breast cancer. Attenuation of this response may represent a previously unrecognized contribution of FGFR1 to tamoxifen resistance.

During breast tumor progression, the transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step with large implications for prognosis. However, the mechanisms of invasion are still largely unknown. At the DCIS stage, there is an over-representation of HER2-positive lesions compared with invasive breast cancer. In this study, we investigated the associations between gene expression profiles in cancer cells and the immune microenvironment of HER2-positive DCIS and invasive breast tumors with concurrent DCIS using spatial transcriptomics. We found distinctly more B cells in the vicinity of DCIS ducts than in invasive tumor areas. There was higher expression of genes involved in energy metabolism in DCIS cancer cells than in invasive cancer cells and a positive correlation between expression of metabolic genes and B-cell abundance in DCIS. In contrast were processes related to epithelial to mesenchymal transition negatively correlated with B-cell abundance in DCIS. We also found significant correlation between expression of the B-cell-attracting chemokines CCL19, CCL21 and CXCL13 in stromal cells and B cell abundance in DCIS. This study indicates that B cells may play a protective role in the progression of HER2-positive DCIS to invasive breast cancer and that increased metabolic activity in intraductal cancer cells in combination with chemokines produced by stromal cells may influence the immune microenvironment of DCIS. These findings have implications for understanding HER2-positive breast cancer progression.

FOXA1 is a key transcription factor that mediates the effects of estrogen receptor (ER) and HER2 signaling in breast cancer. However, the mechanisms underlying FOXA1 regulation by HER2 and ER remain poorly understood. Here, we investigated FOXA1 regulation in cells with varying HER2 levels and its impact on endocrine therapy response. Chromatin interaction analyses revealed that high HER2 levels enhance FOXA1 binding to chromatin regions while reducing ER occupancy. Mechanistically, FOXA1 is acetylated by the histone acetyltransferase EP300 at the WD1 domain in ER-positive cells, attenuating its DNA binding at HER2-induced chromatin regions. Conversely, FOXA1 deacetylation-triggered by HER2/HER3 activation-increases its binding to ER-independent regions and promotes insensitivity to hormone therapy. In a luminal breast cancer patient-derived xenograft model, HER2/HER3 signaling increased FOXA1 chromatin binding and reduced sensitivity to ER-targeted treatment. We identify HDAC2 as a key deacetylase modulating FOXA1 acetylation and partially mediating the effects of HER2/HER3 signaling. Altogether, our findings highlight the significance of FOXA1 acetylation, regulated by the HER2/HER3-HDAC2-FOXA1 axis, in controlling FOXA1 chromatin binding and shaping breast cancer progression and therapy response. These insights may inform future therapeutic strategies.

"A Guide to Breast Cancer Research: From Cells and Molecular Mechanisms to Therapy" is designed as a comprehensive reference for early career investigators and postgraduate students. This book aims to provide a broad overview of contemporary breast cancer research. It covers key areas including development and cancer, metastasis and immunology, subtypes, signalling, therapy, and resistance. This book is organised into seven sections addressing mammary gland development, model systems, cellular origins and heterogeneity, cellular and molecular bases, signalling pathways, metastasis and immunity, and treatment and resistance mechanisms. A few topics such as specific signalling pathways, some emerging therapies, imaging technologies, and AI applications are only briefly mentioned or omitted, reflecting the ever-evolving nature of breast cancer research. This book emphasises the importance of collaboration in advancing cancer research. Initiatives like the Cancer Moonshot and Cancer Grand Challenges advocate for "radical collaboration" of researchers with shared visions and resources. We also note the significance of global efforts in breast cancer research, the need for addressing disparities in care across different regions and for equity in healthcare. Overall, this book showcases milestones and advances in breast cancer research over the past three decades, reflecting significant progress in understanding and treating the disease, which has led to improved patient outcomes.

Alterations in the receptor tyrosine kinase Fibroblast Growth Factor Receptor 1 (FGFR1), such as gene amplification and overexpression, are frequently observed in hormone receptor-positive breast cancers, driving aberrant signaling pathways that promote tumor growth, metastasis, drug resistance, and immune evasion. This study investigates the dual role of FGFR1 in mediating endocrine resistance and modulating innate immune sensing via the cGAS-STING pathway and Type 1 interferon (IFN) responses. Our findings reveal a direct link between FGFR1 activity and endocrine resistance in estrogen receptor-positive T47D breast cancer cells. Strikingly, FGFR1 expression negatively correlates with Type 1 IFN responses in these cells, while endocrine-resistant cells exhibit upregulated STING expression at both mRNA and protein levels. Pharmacological inhibition of FGFR1 using erdafitinib or shRNA-mediated silencing enhances the expression of interferon-stimulated genes (ISGs) by activating the STING-TBK1-IRF3 signaling axis. Mechanistically, we identified that constitutive FGFR1 kinase activity activates AKT, which suppresses STING-TBK1-mediated Type 1 IFN responses as a negative feedback mechanism. In vivo, the combination of FGFR1 inhibition endocrine therapy led to tumor regression in a luminal patient-derived xenograft (PDX) model, driven by an enhanced tumor-intrinsic Type 1 IFN response. These results suggest that FGFR1 acts as an innate immune checkpoint, enabling tumor cells to suppress STING mediated Type 1 IFN response and evade antitumor immunity in hormone receptor-positive breast cancer. Targeting FGFR1 may, therefore, represent a promising strategy to overcome endocrine resistance and restore immune surveillance in this context. Sushil Dhakal, Torbjørn Amundsen Lien, Phuong Vu, Helene Midtun Flatekvål, Jens Henrik Norum, Therese Sørlie. FGFR1 promotes endocrine resistance and regulates DNA sensing via STING modulation in hormone receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6141.

OBJECTIVE:At present, no reliable blood-based biomarkers have been established for patients with endometrial cancer. Liquid biopsies, which can detect circulating tumor DNA (ctDNA), provide a non-invasive way to assess prognosis, monitor tumor evolution and treatment response. We aimed to examine the feasibility and performance of ctDNA as a prognostic tool in a multi-center cohort of EC patients with matched tumor samples. METHODS:Blood plasma samples were collected preoperatively from 83 patients at three European cancer centers. Circulating cell-free DNA (cfDNA) was isolated and analyzed using the Oncomine™ Pan-Cancer cell-free assay. Tumor tissue from 56 of the 83 patients was subjected to whole-exome sequencing, and clinical data were collected for oncological outcome assessment. RESULTS:The mean input of cfDNA was 8.17 ng (range 1.47-29.12 ng). Sixteen (19.3 %) patients were considered ctDNA positive with mutations in one or more genes. Most alterations detected in plasma were concordant with mutations found in the matched tumor for the paired cases. The preoperative presence of ctDNA was associated with a significantly higher rate of recurrence (37.5 % vs 11.9 %, P = 0.024). Although eight of the 14 (57 %) patients with recurrence were negative for ctDNA at diagnosis, positive ctDNA status remained an independent predictor of recurrence also when controlling for other known histopathologic risk factors (HR 5.49, 95 % CI 1.5-20, P = 0.010). CONCLUSIONS:Our results demonstrated the feasibility of using an off-the-shelf gene panel to detect ctDNA in patients with endometrial cancer. ctDNA positivity was significantly associated with worse oncological outcomes.

The objective of the present study was to characterize the molecular features of endometrial carcinomas with ambiguous histology. Eighteen carcinomas that could not be conclusively typed based on morphology and immunohistochemistry underwent analysis of mismatch repair (MMR) status, microsatellite status, and whole-exome sequencing. None of the tumors had pathogenic POLE mutation. Twelve tumors (67%) were microsatellite stable, and 6 (33%) had microsatellite instability. Fourteen tumors (78%) harbored TP53 mutations, and 2 (11%) had mutations in MMR genes. Eleven carcinomas (61%) were classified as copy number high and 7 (39%) as MSI-hypermutated, the latter including 3 tumors with TP53 mutation who concomitantly had MSI or mutation in a MMR gene. Other mutations that were found in > 1 tumor affected MUC16 (7 tumors), PIK3CA (6 tumors), PPP2R1A (6 tumors), ARID1A (5 tumors), PTEN (5 tumors), FAT1 (4 tumors), FAT4 (3 tumors), BRCA2 (2 tumors), ERBB2 (2 tumors), FBXW7 (2 tumors), MET (2 tumors), MTOR (2 tumors), JAK1 (2 tumors), and CSMD3 (2 tumors). At the last follow-up (median = 68.6 months), 8 patients had no evidence of disease, 1 patient was alive with disease, 8 patients were dead of disease, and 1 patient died of other cause. In conclusion, based on this series, the molecular landscape of endometrial carcinomas with ambiguous histology is dominated by TP53 mutations and the absence of POLE mutations, with heterogeneous molecular profile with respect to other genes. A high proportion of these tumors is clinically aggressive.

Abstract Up to 50% of patients diagnosed with ductal carcinoma in situ (DCIS) never experience progression to invasive disease, even if left untreated. Current knowledge of what makes DCIS become invasive is limited and we lack diagnostic tools to predict which patients can be spared treatment. Escape of tumor cells from the breast ducts is influenced by characteristics of the tumor cells, the microenvironment surrounding the ducts, and the interplay between the two. Intraductal tumor cells in DCIS are not physically in contact with extraductal stromal cells. Until now, analyses of DCIS tissue have mainly been performed on bulk tissue, however, this approach does not take into consideration the unique morphology of DCIS. We used the Nanostring GeoMX® digital spatial profiling platform to determine the transcriptome of DCIS tumor cells and the surrounding stromal cells separately. From a large cohort of >500 DCIS cases from Akershus and Oslo University Hospitals, we selected 23 pure DCIS cases, grade 3, of different molecular subtypes: triple negative (TN), Luminal A (LumA) and HER2-enriched. At least four tumor-stroma pairs were selected from each case. Bioinformatic analyses were used to explore both tumor and stroma expression data and the interplay between the two cellular compartments. There were distinct gene expression differences between DCIS of different subtypes. Intertumoral heterogeneity was larger than intratumoral heterogeneity in all subtypes. This was also apparent in stromal cell compartments, although less pronounced. Gene ontology analyses of DCIS tumor cells from each of the three subtypes showed several overlapping biological processes, suggesting common mechanisms for regulating tumor cell growth in DCIS. We also found subtype specific differences: Tumor cells of the TN subtype showed upregulation of protein synthesis. Processes associated with RNA splicing were upregulated in tumor cells of the LumA subtype, whereas the HER2-enriched subtype showed increased DNA repair activity. The immune microenvironment differed between the subtypes: LumA DCIS were characterized by lower immune cell infiltration than TN and HER2-enriched, however, there was variation between and within the cases. Using in silico cell deconvolution, we found that B-cells were more abundant in HER2-enriched tumors, while T-cells were more common in TN and LumA. T-regulatory cells were found in all subtypes. The GeoMX® digital spatial profiling platform is well suited for exploring the transcriptome of DCIS samples. We found biologically relevant differences between tumor cells of the different molecular subtypes already at the DCIS stage in breast cancer progression. The immune cell composition surrounding DCIS lesions also differed between the subtypes. Further studies of the tumor-stroma interplay are required to understand the processes involved in progression of DCIS to invasive disease. Citation Format: Helga Bergholtz, Jens Henrik Norum, Tonje G Lien, Hossein Schandiz, Torill Sauer, Jürgen Geisler, Therese Sørlie. Spatial transcriptomics of ductal carcinoma in situ reveal subtype specific differences in tumoral and stromal cell compartments [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR05.

Objective: There is scarce real-world evidence on patients with advanced/recurrent endometrial cancer treated with platinum-based chemotherapy. We assessed the oncological outcome in groups by molecular classification. Methods: This retrospective cohort study included patients with advanced/recurrent endometrial cancer treated with platinum-based chemotherapy after hysterectomy at The Norwegian Radium Hospital, Oslo University Hospital, Norway, between January 2006 and December 2017. Patients were molecularly classified as pathogenic POLE mutated, mismatch repair deficient, p53 abnormal, or no specific molecular profile. Time-to-recurrence and cancer-specific survival were calculated. Results: We identified 264 advanced-stage patients (stage III/IV) and 96 patients with recurrent disease. The molecular classification was prognostic for time-to-recurrence (p < .0001) and cancer-specific survival (p < .0001) in patients with advanced disease, but the outcome did not differ significantly by molecular groups in recurrent patients. In all molecular groups, patients with stage III disease had longer time-to-recurrence and cancer-specific survival compared to patients with stage IV disease. The worst outcome was observed in patients with p53 abnormal tumors with an HR of 1.57 (95% CI 1.07 to 2.30) for time-to-recurrence and HR of 1.78 (95% CI 1.19 to 2.65) for cancer-specific survival in stage III/IV disease and an HR of 1.45 (95% CI 0.83 to 2.52) for time-to-recurrence and HR of 1.60 (95% CI 0.99 to 2.68) for cancer-specific survival in patients with recurrent disease. The few patients with POLE mutated tumors had favorable outcomes despite the advanced/recurrent disease status. Conclusions: Oncological outcomes differ by molecular groups, in particular among patients with advanced disease. Patients with p53 abnormal tumors have the worst outcome, while patients with POLE mutated tumors have favorable outcomes even with recurrent disease. Implementation of the addition of immunotherapy to chemotherapy is expected to lead to substantial improvement of outcome, particularly in patients with mismatch repair deficient advanced/recurrent disease. There is still a high unmet need in advanced/recurrent patients with p53 abnormal and no specific molecular profile tumors.

BackgroundEMIT-1 is a national, observational, single-arm trial designed to assess the value of the Prosigna, Prediction Analysis of Microarray using the 50 gene classifier (PAM50)/Risk of Recurrence (ROR), test as a routine diagnostic tool, examining its impact on adjuvant treatment decisions, clinical outcomes, side-effects and cost-effectiveness. Here we present the impact on treatment decisions.Patients and methodsPatients with hormone receptor-positive, human epidermal growth factor receptor 2-negative pT1-pT2 lymph node-negative early breast cancer (EBC) were included. The Prosigna test and standard histopathology assessments were carried out. Clinicians’ treatment decisions were recorded before (pre-Prosigna) and after (post-Prosigna) the Prosigna test results were disclosed.ResultsOf 2217 patients included, 2178 had conclusive Prosigna results. The pre-Prosigna treatment decisions were: no systemic treatment (NT) in 27% of patients, endocrine treatment alone (ET) in 38% and chemotherapy (CT) followed by ET (CT + ET) in 35%. Post-Prosigna treatment decisions were 25% NT, 51% ET and 24% CT + ET, respectively. Adjuvant treatment changed in 28% of patients, including 21% change in CT use. Among patients assigned to CT + ET pre-Prosigna, 45% were de-escalated to ET post-Prosigna. Of patients assigned to ET, 12% were escalated to CT + ET and 8% were de-escalated to NT; of those assigned to NT, 18% were escalated to ET/CT + ET. CT was more frequently recommended for patients aged ≤50 years. In the subgroup with pT1c-pT2 G2 and intermediate Ki67 (0.5-1.5× local laboratory median Ki67 score), the pre-Prosigna CT treatment decision varied widely across hospitals (3%-51%). Post-Prosigna, the variability of CT use was markedly reduced (8%-24%). The correlation between Ki67 and ROR score within this subgroup was poor (r = 0.25-0.39). The median ROR score increased by increasing histological grade, but the ROR score ranges were wide (for G1 0-79, G2 0-90, G3 16-94).ConclusionThe Prosigna test result changed adjuvant treatment decisions in all EBC clinical risk groups, markedly decreased the CT use for patients categorized as higher clinical risk pre-Prosigna and reduced treatment decision discrepancies between hospitals.

Introduction/Background Analysis of circulating tumour DNA (ctDNA) is a promising non-invasive technique that may serve as a diagnostic, prognostic, and disease-monitoring marker. Aim of this study is to investigate the association of ctDNA with clinicopathological characteristics and oncological outcome in endometrial cancer patients. Methodology Blood plasma samples were collected pre-operatively from 83 patients diagnosed with endometrial cancer and treated at the Bern University Hospital, Switzerland (n= 36), Oslo University Hospital (n= 34) and Haukeland University Hospital (n= 13), Norway. cfDNA was isolated and selected mutations were detected using the Oncomine™ Pan-Cancer cell free assay. Comparative analysis of whole exome sequencing data from tumour tissue was performed. Results Baseline clinicopathological data and their association with ctDNA status are provided in table 1. Mean concentration of cfDNA was 11.4 ng/ml. 16 (19.3%) patients were considered ctDNA positive with at least one mutation found in plasma. Mutations in ctDNA were found for the following genes: TP53, ESR1, CTNNB1, PIK3CA, KRAS, NRAS, GNAS, and CCND3. Most mutations were concordant between the solid and liquid biopsies. Interestingly, although 52% of patients presented mutations in PTEN in their primary tumour, the same alteration was not found in the ctDNA. Mean follow-up was 45.7 months. Patients with positive ctDNA presented with a significantly shorter recurrence-free (P= .048) and disease-specific (P= .014) survival compared to ctDNA negative patients. In multivariable Cox regression analysis including stage, histological subtype, grading, and lymphovascular space invasion, ctDNA positivity remained an independent predictor of recurrence (HR 6.0, 95% CI 1.7 – 21.4, p= .009) and disease-specific death (HR 19.4, 95% CI 2.7 – 139.7, p= .004). Conclusion This study shows the feasibility of using a gene panel to identify ctDNA positive patients which will facilitate its use on a larger scale. ctDNA positivity was significantly associated with worse oncological outcome. Disclosures No conflicts of interests related to this work. This study was supported by grants from Swiss National Science Foundation, Foundation for clinical-experimental cancer research, SAKK/ Dr. Paul Jannsen Fellowship, Rakel og Otto Kristian Bruun's Legat.