BACKGROUND:Evaluating human epidermal growth factor receptor 2 (HER2) expression is essential for guiding targeted therapies in breast cancer (BC). In Spain, the CONFIRMA-HER2 registry is the main source of information on HER2 testing in BC. The aim of this analysis was to describe the HER2 status in BC and how it has evolved over the 10 years of the CONFIRMA-HER2 registry. MATERIALS AND METHODS:A retrospective observational study of HER2 testing (2013-2023) was conducted in BC samples from 160 Spanish hospitals, recorded in the CONFIRMA-HER2 registry. The analysis assessed rates and performance of immunohistochemistry (IHC) and in situ hybridization (ISH), as well as assessing changes in HER2 positivity over time. RESULTS:A total of 199,934 HER2 determinations recorded in the CONFIRMA HER2 registry were analyzed. IHC results showed 35.5% IHC 0, 27.8% IHC 1+, 22.7% IHC 2+, 11.2% IHC 3+, and 2.9% invalid/not evaluable. ISH was performed in 26.4% of cases, with 82.1% showing nonamplified HER2 gene status. Overall, 15.0% were HER2-positive, 83.2% HER2-negative, and 1.8% invalid/not assessable. Among HER2-negative cases, 54.9% were classified as HER2-low. Over time, HER2 positivity rates varied, likely reflecting changes in clinical practice influenced by updated guidelines. CONCLUSION:To our knowledge, this study is the first to compile and analyze the largest amount of real-world HER2 testing data in Spain. The CONFIRMA-HER2 registry is a key tool that provides comprehensive information on HER2 status as determined by IHC and ISH.

To validate the predictive value of Total Tumor Load (TTL), based on Cytokeratin 19 (CK19) mRNA copy number measured by OSNA in sentinel lymph nodes (SLNs), and to establish an optimal cut-off for identifying HR + /HER2– breast cancer (BC) patients at high risk of ≥ 4 metastatic lymph nodes (LNs), eligible for adjuvant abemaciclib therapy. A retrospective, international, multicenter study pooling data from six studies conducted between 2000 and 2016 in adult BC patients with SLN biopsies analyzed by OSNA. Variables were TTL (CK19 mRNA copies/µL) in SLNs, number of LNs removed, and number of metastatic ones. We constructed receiver operating characteristics (ROC) curves to determine optimal TTL values and multivariate logistic regression models to identify relevant prognostic factors for predicting ≥ 4 LN metastases. We included 3804 patients with HR + /HER2- tumors of any histological type, complete axillary LN dissection, and > 4 LNs removed; 608 (16.0

Background: The HER2DX assay, a 27-gene test, was developed to provide prognostic information and predict treatment responses in patients with early-stage HER2+ breast cancer. This assay evaluates four gene expression signatures: immune/immunoglobulin (IGG), proliferation, luminal, and HER2, offering a comprehensive risk score, a likelihood estimates for pathological complete response (pCR) and ERBB2 expression levels. In this study, we evaluated the HER2DX assay in HER2+ DCIS, aiming to understand its biology and relationship with HER2+ invasive breast cancer. Methods: Standardized HER2DX genomic test was evaluated centrally on 28 formalin-fixed paraffin-embedded tumor samples of HER2+ DCIS across three hospitals in Spain. DCIS features such as nuclear grade, comedonecrosis, architectural pattern, tumor size and hormone receptor (HR) status were evaluated. Percentage (%) of stromal tumor infiltrating lymphocytes (TILs), their spatial distribution (i.e.: inflamed, desert or excluded), and presence of tertiary lymphoid structures (TLS, defined as spatially organized, non-encapsulated areas of immune cell aggregates with or without germinal center [GC]) were assessed on hematoxylin eosin slides. Descriptive statistics were used. Results: The HER2DX assay was evaluated in 28 cases of HER2+ DCIS. Most cases had a 3 nuclear grade (67.9%) and presented with comedonecrosis (60.7%). HR positivity (HR+) was found in 42.9% of cases, as determined by immunohistochemistry. The median tumor size was 30 mm, with a range from 5 mm to 90 mm. The median % of TILs was 20% (range 0.5-70%). TILs, as a continuous variable, showed a moderate correlation with the HER2DX IGG signature (Pearson correlation coefficient=0.43, p-value=0.023), and a tendency with the inflamed spatial distribution (p-value=0.063). TLS were identified in 82.1% of DCIS samples, and their presence was associated with higher expression of the IGG signature (52.2% TLS in IGG-high vs. 21.7% in IGG-low). Regarding the HER2DX luminal signature, 41.7% of cases were classified as luminal-high, associated with HR+ disease (p=0.025). For the HER2DX proliferation signature, 82.1% of cases were identified as proliferation-low. In HER2DX risk stratification, 100% of cases were categorized as low-risk, 46.4% were pCR-high, and 78.6% were ERBB2-high. Notably, only one (3.6%) HER2+ DCIS case was found to be ERBB2-low. Conclusions: The HER2DX assay revealed that all HER2+ DCIS cases were categorized as low-risk, with most cases showing high ERBB2 expression and a high predicted response to anti-HER2-based therapy. These findings underscore the underlying biology of HER2+ DCIS and its tumor immune microenvironment, indicating that HER2+ DCIS shares similar tumor biology with low-risk HER2+ breast cancer. Citation Format: Esther Sanfeliu, Anabel Martinez-Romero, Mercedes Marín-Aguilera, Vicente Marco, Felip Garcia, Vicente Peg, Blanca González-Farré, Ivonne Vazquez, Patricia Galván, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Angela Aguirre, Laia Paré, Guillermo Villacampa, Antonio Martínez, Jesus Soberino, Aleix Prat, Fara Brasó-Maristany. HER2DX assay in HER2-positive (HER2+) breast ductal carcinoma in situ (DCIS) [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 P5-07-18.

Analytical validation of the VHIO-YWBC protocol

Analytical validation of the VHIO-YWBC protocol for NGS

Complete information of the Case cohort, with clinical features and results from the somatic mutation analysis

Breast milk charactherization as liquid biopsy specim and comparison to plasma

Diagnostic images of the PPBC from case #15 right breast

AURORA NGS panel gene list.

VHIO-YWBC NGS panel gene list

Human epidermal growth factor receptor 2 (HER2) in breast cancer (BC) is an important biomarker for a better decision making of anti-HER2 therapy. Trastuzumab has been the standard of care treatment for HER2 positive patients, but recently new antibody-drug conjugates (ADCs) have given an alternative option in clinical practice. Since ADCs efficacy is due to HER2 antibody localization and internalization in the cell, we hypothesize that cellular sensitivity to the drug may be linked to cellular vesicle trafficking systems and membrane protrusions which indicate the resistance of HER2 internalization. Super resolution fluorescence microscope named HM-1000 achieves a resolution less than 20 nm. With this new tool, we aim to study the intracellular distribution pattern of HER2 including vesicle-like structures and membrane protrusions to better identify responders of anti-HER2 therapies, especially ADCs. A retrospective unicentric study is designed to recruit BC patients who have received ADCs treatment that have formalin fixed paraffin-embedded tissue samples stored for routine diagnosis. 5 baseline tumor samples were stained by commercialized HER2 in situ immunofluorescence, and O2 scavenging buffer was applied and sealed to capture the fluorescent blinking every 30 milliseconds by HM-1000. The 50, 000 image frames per microscopic field were processed using ImageJ software and created super-resolution images. We observed that HER2 signals from ADCs responding patients exhibit predominant membrane localization, especially in those with 3+ immunohistochemistry staining status, compared to ADCs non-responding HER2 3+ cases which show low membrane localization of HER2 and more intracellular distribution. Additionally, this finding is highlighted by intense membrane protrusions in HER2 3+ tumor which present very good response of ADCs. We found different HER2 localization in ADC responding and non-responding patients. The presence of membrane protrusions may facilitate HER2 targeted antibody binding and induce both cellular cytotoxicity from immune effector cells and endocytosis of cytotoxic drug, which explains ADCs response. Further studies are needed to support this finding and better predict the effectiveness of ADCs therapies in HER2 positive tumors. Vicente Peg, Yuko Saruta, Yun Li, Santiago Ramon y Cajal. HER2 protein localization in super-resolution and anti-HER2 therapy response in 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 2472.

List of oligos and probes for each ddPCR assay

Table 1. Samples analyzed by NGS Table 2. VHIO-300 results from solid tumor Table 3. VHIO-YWBC results from BM positive for ctDNA

IntroductionThe analysis of tumor-infiltrating lymphocytes often requires techniques that expand their numbers, potentially introducing bias. To address this, we performed a detailed analysis of minimally cultured TILs to evaluate whether this approach better preserves their characteristics.MethodsThe TIL culture method was based solely on tumor tissue with low IL-2 supplementation to minimize artificial alterations. The validity of this approach was confirmed by the correlation between CD3+ T cell percentages in cultures and infiltration patterns observed by immunohistochemistry. Immunophenotyping, cytokine release, and TCR repertoire analysis were used to characterize CD4+ and CD8+ T cell subsets and their molecular features during minimal expansions.ResultsHigh TIL infiltration areas did not consistently correspond to an increased presence of any T cell subset; both CD4+ and CD8+ T cells frequently coexisted in these regions. In contrast, low TIL infiltration sections often displayed a higher proportion of CD4+ T cells. An inverse correlation between CD4+ T cell percentages and cytotoxic molecules was observed, indicating reduced cytotoxic activity in low-TIL sections with abundant CD4+ T cells. TCR repertoire analysis revealed differences between T cell subsets: CD4+ T cells were associated with longer TRA CDR3 nt and shorter TRB N(D)N nt lengths, along with lower diversity, while CD8+ T cells did not exhibit significant correlation with any TCR feature.DiscussionThis study highlights the distinct biological features of CD4⁺ and CD8⁺ TIL populations within the tumor microenvironment that can be preserved using a minimally expanded TIL approach. The observed associations between IHC patterns, T cell subset composition, cytotoxic potential, and TCR repertoire diversity help identify which biopsy regions yield TILs with greater therapeutic potential, thus providing guidance for TIL selection in immunotherapy.

Sensitivity of the BM ctDNA detection assays.

Trastuzumab deruxtecan showed improved efficacy compared with the treatment of the physician’s choice in human epidermal growth factor receptor 2 (HER2)-low (immunohistochemistry [IHC] 1+ or IHC 2+/in situ hybridization-negative) metastatic breast cancer (BC) patients in the DESTINY-Breast04 and -06 phase 3 clinical trials. Both trials used the Ventana PATHWAY HER2/neu (4B5) IHC assay (PATHWAY 4B5) to select patients. The variety of HER2 IHC assays in clinical use complicates real-world differentiation between HER2-low and HER2 IHC 0. This study assessed concordance between PATHWAY 4B5 and comparator assays in identifying HER2-low samples. Fifty clinical BC samples from a cohort of 300 were stained using PATHWAY 4B5 and centrally scored, according to the American Society of Clinical Oncology-College of American Pathologists 2018 guidelines, as HER2 IHC 0, 1+, 2+, and 3+. Unstained samples were sent to participating laboratories in North and South America, Europe, and the Asia-Pacific regions that were actively scoring HER2 IHC for BC, had 2 independent pathologists, and did not routinely use PATHWAY 4B5 following the CDx protocol. Pathologists stained and scored the samples using their laboratory’s routine protocols. Following virtual alignment on interpretation of HER2 IHC scoring guidelines (postalignment), pathologists were directed to rescore the samples. Pathologist scores were compared with centrally assessed scores; the primary endpoint was positive percentage agreement (PPA) and negative percentage agreement (NPA) for HER2-low versus HER2 IHC 0 based on postalignment scores. Overall, 129 pathologists from 68 laboratories submitted 6270 postalignment scores for analysis. PPA (agreement in identifying HER2-low) and NPA (agreement in identifying HER2 IHC 0) were 84.8% (95% CI, 83.6%-86.0%) and 69.2% (95% CI, 67.0%-71.2%), respectively. Across assay types, postalignment PPA ranged from 61.6% to 95.5% and postalignment NPA ranged from 36.9% to 81.7%. The variation in concordance rates observed between assays suggests assay choice may be important for the correct identification of patients with low levels of HER2-expression who may benefit from HER2-targeted therapies.