Abstract Background. Neoadjuvant endocrine treatment (NET) is used for locally advanced hormone receptor (HR)-positive, HER-2-negative breast cancer in highly selected patients. Aromatase inhibitors (AI) are the preferred option in this setting. Although NET has been proposed to be similarly effective as neoadjuvant chemotherapy (NCT) for certain patients, there are no reliable markers currently available to guide post-surgery decisions. Changes in Ki67 levels and results provided by multi-gene arrays (MGA) before and after neoadjuvant treatment have been suggested to be promising and clinically relevant markers for further decision making. Methods. The NEOLETEXE trial was an open-label, intrapatient cross-over trial including patients with locally advanced HR-positive, HER2-negative breast cancer. Patients were randomized 1:1 to NET with either letrozole or exemestane for 3 months, followed by an intrapatient cross-over to the alternative treatment for another 3 months. Extensive biobanking was performed at multiple time points before and during neoadjuvant therapy. In a subset of patients, gene expression profiling was performed using the Prosigna® (PAM50) assay on both the diagnostic biopsy and the final surgical specimen. Immunohistochemical Ki67 expression was assessed in core biopsies obtained at baseline and in excision specimens collected at the time of surgery. Results. A total of 84 patients were included in the intention-to-treat analysis. The median age was 76 years. Pathological complete responses (pCR) occurred in 6% (n=5). The median follow-up time was 6,3 years. Only nine patients (10,7%) relapsed during the follow-up period. An analysis of Ki67 at baseline (Ki67b) and at the time of surgery (Ki67s) was performed, with levels of Ki67 10% or higher classified as Ki67high. Kaplan-Meier analysis showed that patients with low Ki67 levels at surgery (<10%) experienced significantly better recurrence-free survival (RFS) compared to the Ki67 high group (HR 0,07, CI 0.02-0.31, p < 0.001).Prosigna testing was performed at baseline and at the time of surgery in a cohort of 20 patients. At baseline, 35% (n = 7) of the Prosigna cohort were classified as Luminal A, 57,9% (n = 11) as Luminal B, and one patient was identified as HER2-enriched. At the time of surgery, most tumors were classified as Luminal A (80%, n = 16), two patients were categorized as HER2-enriched, and only one patient remained in the Luminal B category. Conclusions. Our trial strongly underlines that NET involving AI as monotherapy for locally advanced HR-positive breast cancer is a pragmatic and effective alternative to NCT in highly selected patients. Ki67 expression data and MGA data at surgery turned out to be promising markers to potentially guide post-neoadjuvant decision-making and should be tested in future clinical trials. Citation Format: Kamilla Fjermeros, Julius Johannes Hettich, Stephanie Beate Geisler, Unn-Cathrin Buvarp, Hilde Presterud Ødegård, Elin Edda Seland Agustsdottir, Laurens Cornelus Reitsma, Nazli Bahrami, Vessela N. Kristensen, Xavier Tekpli, Torben Lüders, Andliena Tahiri, Manouchehr Seyedzadeh, Torill Sauer, Silje Mathiassen, Sofie Flovik Ranestad, Clara Hammarstrøm, Jürgen Geisler. Neoadjuvant treatment of ER-positive/HER2-negative breast cancer with aromatase inhibitors in sequence: Ki67 dynamics and biology shifts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2288.

Abstract Background: The expression of PD-L1 in tumor-infiltrating lymphocytes (TILs) in tumor microenvironment (TME) of high-grade breast DCIS is currently unknown. Methods: Formalin-fixed paraffin-embedded specimens from 494 female patients diagnosed with DCIS between 1996-2018 were available to routine diagnostic immunohistochemical (IHC) staining. ER and PR IHC positivity was defined as ≥1% positive tumor cells, and HER2 IHC was scored based on the updated guidelines of the American Society of Clinical Oncology and the College of American Pathologists, as in routine diagnostic procedures. HER2 silver in situ hybridization was performed when the IHC score was 2+. We calculated the Ki67 ratio by counting 200 intraductal epithelial cells in two separate hotspot foci. DCIS cases were classified as Luminal A (LumA), LumB HER2ˉ, LumB HER2+, HER2-enriched, or triple-negative (TPN) subtypes according to the 2013 St. Gallen guidelines, which is used for molecular subtyping of invasive breast carcinoma. Each subtype was sorted into “Pure”: without an invasive component and “W/invasive”: with an invasive component. We calculated the PD-L1 IHC ratio by counting (up to) 1000 immune cells divided by positive PD-L1 cells. PD-L1 expression was dichotomized at a threshold of 1%. We assessed the associations between PD-L1 status, subtype, and other variables, including age, Ki67, DCIS extension, and invasiveness. Results: We identified a significant proportion of TILs in 149/484 (31%) cases. 100% of these cases were high-grade DCIS. We successfully stained 118/149 cases with PD-L1 IHC, and 73/118 (63%) expressed PD-L1 ≥1%. In 92/149 (62%) cases with TILs, strong membrane-positive HER2 overexpression was observed (25% LumB HER2+ and 37% HER2-enriched subtype, p < 0.0001). A significant number (31/60; 52%) of the “W/invasive” cases contained TILs, compared to “Pure” (118/362; 32%) (p < 0.0055). PD-L1 ≥1% was most prevalent in HER2-enriched (48.6%) and LumB HER2+ (25%) subtypes, with lower representation in LumA (12.5%), LumB HER2ˉ (9.7%), and TPN (4.2%) subtypes. However, comparison of the PD-L1 ≥1% status across subtypes yielded a non-significant chi-square result (p = 0.2734). PD-L1 expression was not significantly associated with “W/invasive” cases (p = 0.0765). Significant differences were observed across subtypes among cases expressing PD-L1 ≥1% for Ki67 (p = 0.0325) and DCIS extension (p = 0.0323), but not for age (p = 0.4329) or invasive status (p = 0.6722). Conclusions: PD-L1 ≥1% was more common in HER2-driven subtypes, but its distribution was not subtype-dependent. PD-L1 expression also lacked a significant association with invasiveness. These findings demonstrated strong PD-L1 expression in high-grade DCIS, although the utility of PD-L1 as a standalone biomarker for subtype stratification or prediction of invasive potential in DCIS patients could be limited. Citation Format: Hossein Schandiz, Lorant Farkas, Berit Gravdehaug, Elin Edda Seland Agustsdottir, Torill Sauer, Jürgen Geisler. Programmed cell death ligand 1 (PD-L1) expression across molecular subtypes of human high-grade ductal carcinoma in situ (DCIS) of the breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4977.

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.

Background The aromatase inhibitors (AI) letrozole and exemestane, are often used in sequence in targeting ER+ breast cancers. However resistance to AI poses a major barrier to sustained clinical benefit, while the biological mechanisms underlying the phenomenon remain largely unknown. In this study, we build on our clinical NeoLetExe trial, with the aim to investigate the molecular basis of resistance to AI, by analysing subclonal evolutionary dynamics during sequential treatment. Methods We use whole-exome sequencing (WES) data from 11 ER+ breast cancer patients and 3 timepoints of the Neoletexe trial to reconstruct cancer cell fraction–based subclonal composition. Single-cell DNA sequencing from matched tumour samples is used for validating identified clones and variants. Subclonal variants were annotated to genes by integrating evidence from public data and ExpectoSc. Pathway enrichment analysis using Human Base was conducted. Results Higher cancer cell fraction clone trajectories were significantly associated with reduced treatment response (p = 0.023). Clones reconstructed by WES were validated at 81% using single-cell DNA sequencing. Clones resistant to both letrozole and exemestane demonstrated PIK3CA/AKT/mTOR signaling activation, KRAS pathway dysregulation, hedgehog signaling, and androgen receptor pathways, alongside extensive immune activation and metabolic reprogramming. Drug-specific resistance patterns showed exemestane-resistant clones enriched for epigenetic control and miRNA-mediated silencing, while letrozole-resistant clones displayed metabolic dysregulation but notably lacked immune pathway activation. In contrast, treatment-sensitive clones maintained coordinated cell cycle control, preserved DNA damage responses, and retained immune signaling capacity. Analysis of FDA-approved breast cancer targets identified actionable alterations in PIK3CA (4 patients) and AKT1 (1 patient) that persisted through AI treatment, with RNA expression analysis revealing 48 additional therapeutic targets spanning PI3K/AKT/mTOR, CDK4/6, DNA repair (BRCA1/2, ATM), and immune checkpoint pathways. Conclusion WES-based cancer cell fraction analysis successfully captured subclonal evolutionary trajectories during AI treatment, revealing drug-specific mechanisms and identifying key molecular players in endocrine therapy resistance. This work establishes a framework for precision oncology approaches by providing actionable therapeutic targets and advancing our understanding of resistance mechanisms to improve clinical outcomes in sequential AI therapy. ### Competing Interest Statement The authors have declared no competing interest.

Simple summaryWe investigated the role of Ki67, a ubiquitous marker in cancer, within the context of ductal carcinoma in situ (DCIS), a precursor of invasive breast cancer. Through rigorous analysis of histopathological and immunopathological samples from a substantial cohort, this study revealed robust correlations between heightened Ki67 expression, diminished progesterone (PR) levels, and HER2 overexpression, indicative of aggressive DCIS phenotypes. These findings offer novel insights into the surrogate immunomolecular subtyping landscape of DCIS, potentially refining risk stratification and therapeutic approaches. This elucidation underscores the translational significance of Ki67 as a prognostic and predictive biomarker in DCIS, with implications for personalized treatment paradigms and patient outcomes.BackgroundThe Ki67 proliferation index is widely used in various tumors, including invasive breast carcinoma (IBC). However, its prognostic utility is often constrained by technical complexity. Its diagnostic and clinical significance in ductal carcinoma in situ (DCIS) remains uncertain. We studied Ki67 immunohistochemistry interobserver diagnostic agreement at different cutoff values in high-grade DCIS. Additionally, we investigated the associations between Ki67 expression, PR levels, and human epidermal growth factor receptor 2 (HER2) in high-grade DCIS among various subtypes (Luminal (Lum) A, LumB HER2-, LumB HER2+, HER2-enriched, and triple-negative)).MethodsUsing histopathological specimens from 484 patients diagnosed with DCIS between 1996 and 2018, we implemented the 2013 St. Gallen recommendations for surrogate immunomolecular subtyping of IBC. Subtypes were classified, and the Ki67 interobserver diagnostic agreement between Counting Pathologist 1 (CP1) and CP2 was calculated using Cohen’s kappa coefficient at various cutoff values.ResultsThe Cohen’s kappa coefficient for interobserver agreement between CP1 and CP2 was κ = 0.586, indicating moderate agreement. Ki67 levels varied significantly among subtypes (p < 0.0001), with a median Ki67% being higher in cases with invasive components (p = 0.0351). Low PR combined with high Ki67% was significantly associated with HER2 overexpression (p = 0.0107).ConclusionsInterobserver agreement for the Ki67 count was moderate. Ki67 expression showed considerable variability in high-grade DCIS. Low PR levels combined with high Ki67 expression were linked to HER2 overexpression, showing possible clinical implications for identifying high-risk DCIS.

Background: We investigated the distribution of tumor-infiltrating lymphocytes (TILs) in the extracellular matrix (ECM) of high-grade breast ductal carcinoma in situ (DCIS) among various subtypes. Methods: This study included formalin-fixed, paraffin-embedded specimens from 494 female patients diagnosed with DCIS between 1996 and 2018. Due to reservations received from 10 patients, we excluded these cases from further analysis. Using the Van Nuys classification system, experienced breast pathologists actively graded histopathological specimens. The extent of DCIS was recorded when this information was available. Routine diagnostic immunohistochemical (IHC) staining was performed. ER and PR IHC positivity was defined as > 1% positive tumor cells, in accordance with the updated guidelines of the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). HER2 IHC was scored based on the ASCO and CAP guidelines, as in the routine diagnostic procedures for invasive breast carcinoma (IBC). HER2 silver in situ hybridization was performed when the IHC score was 2+. We calculated the Ki67 ratio by counting 200 intraductal epithelial cells in two separate hotspot foci. DCIS cases were classified as Luminal A (LumA), LumB HER2ˉ, LumB HER2+, HER2-enriched, or triple-negative subtypes according to the 2013 St. Gallen guidelines, which is used for molecular subtyping of IBC. Each subtype was divided into three categories: "Pure", which indicates no invasive component, "W/invasive", which indicates an invasive component, and "All", which represents the entire group of the given subtype. Results: Of the 484 DCIS cases, 422 were identified as high-grade, of which 60 (14%) presented with invasive lesions. A significant number (31 out of 60; 52%) of the “W/invasive” cases contained TILs, compared to those classified as “Pure” (118 out of 362; 32%), (p < 0.0055, Fisher’s exact test). We found a significant portion of TILs in the ECM in 149 of 484 (31 %) cases. All these cases (100%) were classified as high-grade DCIS. We were able to subtype 140 out of 149 of these cases. We found that 66% (n = 92) of high-grade DCIS cases with TILs showed strong membrane positive HER2 overexpression (IHC score 3+). Of these, 27% (n = 37) were LumB HER2+, and 39% (n = 55) were HER2-enriched subtypes (p < 0.0001, Fisher’s exact test). The proportions of LumA was 12% (n = 17), LumB HER2ˉ was 13% (n = 18), and finally TPN was 9% (n = 13), respectively. DCIS extent differed significantly between “Pure” cases and “W/invasive” cases with a median of 20 mm and 30 mm, respectively, (p = 0.0197, Mann-Whitney test) and between those with TILs and those without TILs, with a median of 28 mm and 20 mm, respectively (p = 0.0040, Mann-Whitney test). Differences in age between patients with TILs and those without TILs were not significant, with median ages of 55 and 57 years, respectively (p = 0.2534, Mann-Whitney test). Conclusion: We found that high-grade DCIS cases with an invasive component exclusively showed the presence of TILs in the ECM, highlighting their potential as a tumor progression marker. Notably, a substantial proportion of high-grade DCIS simultaneously exhibited TILs in the ECM and strong HER2 overexpression (IHC score 3+). We found this association to be particularly pronounced in the HER2-enriched subtype (p = 0.0035, Fisher’s exact test). Citation Format: Hossein Schandiz, Lorant Farkas, Daehoon Park, Elin Edda Seland Agustsdottir, Berit Gravdehaug, Yan Liu, Torill Sauer, Jürgen Geisler. The role of tumor-infiltrating lymphocytes (TILs) in the progression of human high-grade ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-02-22.

Abstract Objective: This study aimed to investigate the molecular details in breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 were investigated among luminal A (LumA), luminal B HER2-negative (LumB HER2-), luminal B HER2-positive (LumB HER2+), HER2-enriched, and triple-negative (TPN), subtypes of DCIS. Materials & Methods: The study comprised formalin-fixed paraffin-embedded (FFPE) specimens of 357 DCIS grade 3 cases diagnosed between 1996 – 2018. Routine diagnostic immunohistochemical (IHC) staining were performed. DCIS cases were classified as LumA, LumB HER2-, LumB HER2+, HER2-enriched or TPN, according to the 2013 St. Gallen guidelines, which is used for molecular subtyping of invasive breast carcinoma (IBC). Each subtype was sorted into three subcategories: “pure” meaning those without an invasive component; “w/invasive” meaning those with an invasive component; and “all” meaning the entire group of the given subtype. Furthermore, ER and PR-receptor expression was registered for LumA, LumB HER2- and LumB HER2+ cases as intervals. The distribution of Ki67 was analyzed within each subtype. For some analyses, we combined the LumA and LumB HER2- subtypes into one entire group. The inter-observer variability of Ki67 was calculated by setting a cut-off value of 20% (2013 St. Gallen). A cohort (n = 47) of DCIS cases with a median Ki67 value of 18% was selected. Ki67 was calculated by analyzing 200 DCIS cells in two separate hotspots. Cohen's Kappa coefficient was calculated based on these data. HER2 (IHC) was scored based on ASCO/CAP guidelines established for routine diagnostic work-up for IBC. 16 equivocal cases (2+) were further investigated using dual SISH. Results: 98% of “all” cases of the LumA subtype showed an ER ≥ 50%. PR expression ≥ 50% was found in 91% of cases in this subtype. The incidence of ER-receptor at a cut-off ≥ 50% in the LumA subtype was significantly higher than that in the LumB HER2- and LumB HER2+ subtypes (p < 0.0001, Chi-square). In contrast, there was a statistically significant reduction in ER-receptor expression at a cut-off < 10% in LumA compared to the latter subtypes. The proportion of cases with PR-receptors with a cut-off of < 20% showed significant differences between LumA, LumB HER2- and LumB HER2+ subtypes (1.6%; 47% and 37%, respectively). There was also a significantly higher proportion of PR-receptor ≥ 50% cases among the LumA subtype. We found a significant association between PR < 20% and HER2 (3+) in luminal subtypes (p < 0.0004, Fisher's exact). There was no significant difference in ER/PR expression in “pure” cases of luminal subtypes of DCIS versus those with an invasive component (p = 0.1831, Chi-square). The Ki67 in the entire population (n = 357) varied from < 1% to > 80%. The mean and median were around 20% in those subtypes whose classifications were not depended on Ki67. There was a significant difference in the distribution of Ki67 when cases of LumA “all” and those of LumB HER2- “all” were combined as one entire group and were compared to LumB HER2+ “all” and HER2-enriched “all” (p-adjusted < 0.0001, Kruskal Wallis). The assessment of Ki67 among different observers showed a Cohen's kappa coefficient of 0.29 – 0.31 (fair agreement). We compared the HER2 (IHC) 0, 1+ and 2+ score among LumA and LumB HER2- subtypes and did not find a statically significant difference, when the “pure” and “w/invasive” were compared (p = 0.603, Chi-square). Conclusions: Ki67 was highly variable in DCIS grade 3. Inter-observer agreement was (as expected) suboptimal, and the cut-off at 20% defined by the 2013 St Gallen guidelines for IBC is not reliable for the distinction of LumA and LumB HER2 subtypes of DCIS. The LumB subtype of DCIS is heterogeneous with considerable variability among the four IHC markers used in the present study. A low PR is strongly associated with HER2 (3+), in luminal subtypes (p < 0.0004, Fisher's exact). Citation Format: Hossein Schandiz, Lorant Farkas, Daehoon Park, Solveig Norheim Andersen, Jürgen Geisler, Torill Sauer. Towards personalized medicine for DCIS - the role of hormone receptors, HER2, and Ki67 status in high-grade DCIS [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-10.

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.

Abstract Exemestane, an aromatase inactivator of the third generation, plays a crucial role in breast cancer therapy by targeting the P450 aromatase enzyme and, thus, decreasing estrogen synthesis. Exemestane (Aromasin™) is currently the only steroidal aromatase inactivator widely used in clinical routine treatment of ER+ breast cancer in all phases of the disease in a global perspective. However, the complex mechanisms underlying its therapeutic effects, besides being an aromatase inhibitor, remain incompletely understood. In this study, we employed a combination of human samples and in vitro data to unveil a compelling insight: Exemestane (EXE) and its primary metabolite, 17β-hydroxyexemestane (HEXE), exhibit potent inhibitory effects on tumor growth when present together in patient serum. Our biochemical analysis establishes a critical threshold—20% HEXE metabolite of the total EXE in patient serum—to trigger a tumor growth inhibition exceeding 90%, as evidenced by Ki67 staining. Mechanistically, our data reveals that both HEXE and EXE bind to the Androgen Receptor (AR), triggering a synergistic activation that induces a transcriptional program leading to cell death while diminishing the intracellular signaling activated by the oncogene Ras. Notably, patients with tumors characterized by a minimum of 20% AR+ epithelial cancer cells stand to benefit the most from exemestane and HEXE. Intriguingly, the binding of AR to chromatin in tumors gives rise to a molecular signature capable of distinguishing responsive from non-responsive patients to both EXE and HEXE. Collectively, our findings elucidate a dual therapeutic role for Exemestane in selected patients: it not only restrains estrogen-driven proliferation by estrogen suppression but also stimulates cell death by establishing a specific interactome with the AR at the genomic level. These insights suggest that both EXE and HEXE are necessary to achieve the best therapeutic effects in ER+/AR+ breast cancer tumors. Moreover, our findings suggest that AR-expression and targeting should be investigated further to potentially add novel strategies to our existing algorithms in ER+/AR+ MBC. Citation Format: Gemma Santacana-Font, Darek Kedra, Maria del Carmen García-Macías, Laurens Cornelus-Reitsma, Marianne Lyngra, Torill Sauer, Vessela Kristensen, Antoni Hurtado, Jürgen Geisler. Exemestane and its primary metabolite 17-hydroexemestane inhibit synergically the tumor growth of ER/AR positive breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7576.

The aromatase inhibitor letrozole and the aromatase inactivator exemestane are two of the most pivotal cancer drugs used for endocrine treatment of ER-positive breast cancer in all phases of the disease. Although both drugs inhibit CYP19 (aromatase) and have been used for decades, a direct head-to-head, intra-patient-cross-over comparison of their ability to decrease estrogen synthesis in vivo is still lacking. Postmenopausal breast cancer patients suitable for neoadjuvant endocrine therapy were randomized to receive either letrozole (2.5 mg o.d.) or exemestane (25 mg o.d.) for an initial treatment period, followed by a second treatment period on the alternative drug (intra-patient cross-over study design). Serum levels of estrone (E1), estradiol (E2), letrozole, exemestane, and 17-hydroxyexemestane were quantified simultaneously using a novel, ultrasensitive LC–MS/MS method established in our laboratory. Complete sets of serum samples (baseline and during treatment with letrozole or exemestane) were available from 79 patients, including 40 patients starting with letrozole (cohort 1) and 39 with exemestane (cohort 2). Mean serum estrone and estradiol levels in cohort 1 were 174 pmol/L and 46.4 pmol/L at baseline, respectively. Treatment with letrozole suppressed serum E1 and E2 to a mean value of 0.2 pmol/L and 0.4 pmol/L (P < 0.001). After the cross-over to exemestane, mean serum levels of E1 and E2 increased to 1.4 pmol/L and 0.7 pmol/L, respectively. In cohort 2, baseline mean serum levels of E1 and E2 were 159 and 32.5 pmol/L, respectively. Treatment with exemestane decreased these values to 1.8 pmol/L for E1 and 0.6 pmol/L for E2 (P < 0.001). Following cross-over to letrozole, mean serum levels of E1 and E2 were significantly further reduced to 0.1 pmol/L and 0.4 pmol/L, respectively. Serum drug levels were monitored in all patients throughout the entire treatment and confirmed adherence to the protocol and drug concentrations within the therapeutic range for all patients. Additionally, Ki-67 values decreased significantly during treatment with both aromatase inhibitors, showing a trend toward a stronger suppression in obese women. To the best of our knowledge, we present here for the first time a comprehensive and direct head-to-head, intra-patient-cross-over comparison of the aromatase inhibitor letrozole and the aromatase inactivator exemestane concerning their ability to suppress serum estrogen levels in vivo. All in all, our results clearly demonstrate that letrozole therapy results in a more profound suppression of serum E1 and E2 levels compared to exemestane.

Cystic echinococcosis, although rare in Europe, presents a diagnostic challenge when encountered, especially in atypical locations such as the mesentery. This case report is significant because it highlights the unique presentation of mesenteric hydatid cysts, emphasizing the importance of considering uncommon etiologies in differential diagnosis, particularly in immigrant populations. The novelty of this case lies in its rarity and the diagnostic dilemma it posed, ultimately leading to successful management through prompt recognition and accurate diagnosis. A 33-year-old pregnant female from East Africa presented with intermittent abdominal pain during pregnancy. Imaging revealed a cystic mass adjacent to the mesentery, initially misdiagnosed as an ovarian cyst. Postpartum, she developed acute abdominal symptoms, leading to a revised diagnosis of a ruptured hydatid cyst. Antiparasitic treatment and surgical intervention were initiated, resulting in successful management. This case underscores the necessity of prompt recognition and accurate diagnosis of rare conditions such as mesenteric hydatid cysts, particularly in immigrant populations. A multidisciplinary approach is crucial for optimal patient care in such cases.

Objective:In this study, we investigated pivotal molecular markers in human high-grade breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2) was measured among various subtypes (Luminal (Lum) A, LumB HER2-, LumB HER2+, HER2-enriched and triple-negative). Methods:In total, 357 DCIS cases were classified into respective subtypes, according to the 2013 St. Gallen guidelines. Each subtype was categorized into three subcategories: "Pure" (those without an invasive component), "W/invasive" (those with an invasive component), and "All" (the entire group of the given subtype). ER and PR expression were registered as intervals. Equivocal HER2 immunohistochemistry (IHC) cases (2+) were further investigated using dual-color in situ hybridization. Results:The majority of patients (71%) were over the age of 50. We discovered no significant differences in the proportion of age between the "Pure" and "W/invasive" groups. There was no significant difference in ER/PR expression between "Pure" luminal subtypes of DCIS and "W/invasive" cases. We compared the HER2 IHC scores of "0", "1+", and "2+" among LumA and LumB HER2 subtypes and identified no statistically significant differences between "Pure" and "W/invasive" (p = 0.603). ER and PR expression ≥ 50% cutoff value was present in > 90% of all LumA cases. The incidences of cases with ER expression at cutoff values of < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). The proportion of cases with PR expression < 20% showed significant differences in the various luminal subtypes. In luminal B subtypes, low PR expression (< 20%) was significantly associated with both strong HER2 expression (3+) and the presence of an invasive component (p = 0.0001 and p = 0.0365, respectively). Conclusions:ER and PR expression at ≥ 50% cutoff values were found in more than 90% of LumA cases. Samples with ER < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). Low PR expression in high-grade DCIS was strongly associated with HER2 overexpression (3+) and an invasive component (p = 0.0001 and p = 0.0365, respectively).

Background: Human cytomegalovirus (HCMV) is increasingly suggested to be involved in human carcinogenesis and onco-modulation due to its ability to contribute to all hallmarks of cancer. Growing evidence demonstrates a link between HCMV infection and various malignancies, including breast cancer, which incidence and mortality are still on the rise. The etiology of breast cancer remains mostly unclear, leaving 80% of breast cancer cases considered to be sporadic. Identifying novel risk- and prognostic factors for improved breast cancer treatment and increased survival rates, were the objectives of this study. Methods: Automated immunohistochemical staining results for HCMV proteins in 109 breast tumors and lymph node metastasis were correlated with clinical follow-up data (>10 years). Statistical analyses for median Overall Survival (OS) were performed. Results: Survival analyses revealed shorter median OS for patients with HCMV-IE positive tumors of 118.4 months compared to 202.4 months for HCMV-IE negative tumors. A higher number of HCMV-LA positive cells in the tumors was also associated with a shorter OS in patients (146.2 months vs. 151.5 months). Conclusions: Our findings suggest a link between HCMV-infections and breast cancer prognosis, which paves the way for potential novel clinical intervention and targeted therapy that may prolong the overall survival of selected patients with breast cancer.

Abstract The authors have requested that this preprint be removed from Research Square.

Objective The purpose of this study was to investigate and classify the molecular subtypes of high-grade ductal carcinoma in situ (DCIS) and identify possible high-risk subtypes. The heterogenicity of DCIS with variable clinical and histopathological presentations has been recognized. Nevertheless, only histopathological grading and diameter are currently implemented in clinical decision-making following the diagnosis of DCIS. The molecular subtypes of DCIS and their IHC surrogate markers have not been defined in conventional treatment guidelines and recommendations. We applied the definitions of molecular subtypes according to the IHC surrogate markers defined for IBC and subclassified high-grade DCIS, accordingly. Methods Histopathological specimens were collected, revised, and regraded from 494 patients diagnosed with DCIS between 1996 and 2018. Other in situ and papillary lesions observed in breast biopsies were excluded from this study. 357 high-grade DCIS cases were submitted to IHC analysis. The markers investigated were ER, PR, HER2, and Ki67. Results 45 cases were classified as grade 1, 19 as grade 2, and 430 as grade 3. Sixty patients with high-grade DCIS had an additional invasive component in the surgical specimen. Thirty-three patients were diagnosed with recurrent DCIS or invasive cancer (minimum one year after their primary DCIS diagnosis). The proportions of luminal A and luminal B HER2-negative subtypes varied depending on whether 2011 or 2013 St. Gallen Consensus Conference guidelines were adopted. Luminal A was the most prevalent subtype, according to both classifications. The luminal B HER2-positive subtype was found in 22.1% of cases, HER2-enriched subtype in 21.8%, and TPN subtype in 5.6%. There were strong indications that HER2-enriched subtype was significantly more frequent among DCIS with invasive component ( p = 0.0169). Conclusions High-grade DCIS exhibits all the molecular subtypes previously identified in IBC, but with a somewhat different distribution in our cohort. HER2-enriched subtype is substantially related to the presence of an invasive component in DCIS; consequently, it is regarded as a high-risk entity.

Background: Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous group with variable clinical presentation. 70-80% of DCIS are classified as grade 3 (Van Nuys Classification). They are treated according to grade and extension of the lesion, as was the standard treatment for invasive breast cancer in the 1970`s. Molecular genetic studies of invasive breast carcinomas (BC) have defined a molecular subclassification. In routine diagnostics, we use surrogate markers like estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) as tools to stratify for treatment. The individual results of these surrogate markers have resulted in a complex treatment algorithm. In contrast, still all DCIS grade 3 are given the same treatment. Aim: To investigate the molecular subtypes of DCIS diagnosed at the Department of Pathology, Akershus University Hospital (Norway). Materials: 483 histological specimens of DCIS at Akershus University Hospital, Dep. of Pathology, during 1996-2018. All relevant background information was recorded. Methods: All histological sections have been reassessed, and the grading has been confirmed independently by two experienced pathologists. An eventual peritumoral inflammatory component was made note of. All grade 3 DCIS with representative paraffin blocks were submitted for immunohistochemistry (IHC). The investigated markers were ER, PR, Ki-67 and HER-2. Results: The age ranged from 33-90, with a mean and median of 57 years. 10.4% were grade 1, 3.9% as grade 2 and 85.7% as grade 3. The size of DCIS ranged from 0,5 to150 mm, with mean 28 mm and median of 20 mm. 64 patients had an invasive component, 51 of them with a size of <5 mm (pT1a), and 13 > 5 mm but < 10 mm (pT1b). 33 (6,8%) patients were diagnosed with a recurrent DCIS or cancer (minimum 1year after primary DCIS diagnosis). Six patients were deceased (1 was diagnosed with new ipsilateral DCIS 3 years later, 2 with cancer in the contralateral breast, 2 with cancer in ipsilateral breast and 1 with new DCIS in the ipsilateral breast). The preliminary results of immunohistochemical studies of 245 cases indicate: Luminal A: 30.7% (Ki-67 <14%), Luminal B: 42% (Ki-67> 14%) and non-luminal 27.3%. Around 33% of the non-luminal cases are expected to be triple negative. Histomorphologically, we have found peritumoral lymphoid cells in 31% of cases, and all of these (100%) are DCIS grade 3. Conclusions: The molecular subtypes identified in invasive breast carcinomas are all represented in DCIS G3 and further studies might reveal the possibility of a more adapted treatment algorithm, as in invasive BC. The peritumoral inflammatory cell infiltrates will be investigated in future studies. Citation Format: Hossein Schandiz, Daehoon Park, Yan Liu Kaiser, Marianne Lyngra, Ingrid Solvang Talleraas, Jürgen Geisler, Torill Sauer. A novel diagnostics approach to Ductal Carcinoma In Situ (DCIS) with potential impact on the therapeutic algorithms [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr A015.

Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.