Background: Implant-based postmastectomy breast reconstruction carries a significant risk of complications, including an aproximately 10% rate of implant loss following postmastectomy radiation therapy. While prior radiation, diabetes, and obesity are recognized risk factors, the impact of the quality of skin and soft tissue on these outcomes is not well understood. Currently, no biomarkers are available to guide decisions between implant-based and autologous tissue reconstruction. Mechanical alterations of skin tissue have been shown to be a hallmark of inflammation and thus we hypothesize that detection of this in standard of care samples may provide an early assessment for failure risk. ObjectiveThis study aims to characterize the nanomechanical properties of skin and soft tissue in patients undergoing postmastectomy implant reconstruction, and evaluate correlations between these properties and clinical outcomes, including implant loss and patient-reported quality of life. The overall goal is to detect the nanomechanical changes that are predictive of implant failure in breast cancer mastectomy reconstruction surgery patients. Methods: This data comes from 36 evaluable study subjects from an on-going single center prospective observational study currently taking place in MDACC (NCT06584396), enrolling female patients post- prevention or curative mastectomy, who elected postmastectomy breast reconstruction. Part of the fibrous capsule resection (often containing scar) removed during the breast implantation replacement surgery is analyzed using the ART-1 Nanomechanical Phenotype System, which provides a spatial nanomechanical characterization of clinical samples. For each sample, an average of 15 maps is measured, with 400 indentation points per map, totaling 6000 data points per sample. Clinical and patient reported outcomes are being collected through chart review and a validated BREAST-Q assessment for quality of life. Tissues are further annotated by pathologists and the nanomechanical signature of each sample is correlated to the presence and pathological features of scars. Results and Conclusions: Overall the nanomechanical signature of these samples presents distinguishing features when compared to other studies such as breast cancer and normal breast tissue. While the nanomechanical signature of breast tumor is typically multi-peak and heterogeneous, the stiffness of the skin/scar tissues presents a single tail corresponding to a continuously heterogeneous stroma with stiffer profiles compared to normal breast and breast cancer tissues. The adhesion energy channel also presents a single asymmetric population with a much narrower distribution when compared to both normal breast tissue and breast cancer tissue. The dissipation profile is focused at very low values, whereas both normal breast and breast cancer tissue present much higher dissipation values. As we continue to enroll for this on-going study, we are compiling interim outcome evaluation of surgical outcome. Overall, this provides a baseline characterization of reconstruction tissues which supports the further development of Nanomechanics-based biomarkers for reconstruction failure/success prediction in postmastectomy settings. Citation Format: Papa Diogop Ndiaye, Mark Victor Schaverien, Victor Joseph Hassid, Paul L. Shay, John William Shuck, Mark Warren Clemens, Jessie Zexi Yu, Austin Y Ha, Gregory Reece, Margaret Jane Roubaud, Alexander Francis Mericli, Mark Villa, Ashleigh M. Francis, David Matthew Adelman, Donald Baumann, Joani Marie Christensen, Ryan Dickey, Philip Hanwright, Sahil Kuldip Kapur, Jonathon Bryce Edward Olenczak, Aysegul A Sahin, Edna Paredes, Ahmed Jizawi, Gitika Srivastava, Tia Darwich, Soufian Lasli, Jacqueline Tilley, Tobias Appenzeller, Sara Nizzero, Marko Loparic, Marija Plodinec, Smith,Benjamin. Characterizing Nanomechanical Properties of Skin and Soft Tissue in Postmastectomy Implant Reconstruction [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 P4-04-19.

Abstract Background: We hypothesized that the systemic benefit of regional nodal irradiation (RNI) for breast cancer derives from immune stimulation in involved nodes. We compared involved lymph nodes (ILN) from patients treated on a prospective trial of pre-operative and post-operative conventional (2 Gy/fx) versus hypofractionated (2.67 Gy/fx) RNI regimens (SAPHIRE NCT02912312). Methods: 17 patients ILNs with ER+, pN+ BC were Digitally Spatially Profiled with immune-related proteins. 6 post-op cases had no neoadjuvant chemotherapy (NACT) (untreated controls). All others had NACT +/- RNI. 4 pre-operative RNI short-course (RNI_NACT-2.67Gy/fx); 5 pre-op standard RNI (RNI_NACT-2 Gy/fx); 3 post-op RNI after NACT. 5 Regions of Interest (ROIs) were selected and categorized as “Tumor” (PanCK+) or “Immune” (CD45+) compartments. Cell density and spatial analysis were assessed in selected cases. Pearson’s correlations, T-Test and KS tests were performed on protein expression in all ROIs, FDR < 0.1, P value < 0.05. Signals with Log2 differences between -1 and 1 were analyzed using adjusted LMM for patient-level variation, and PCA of ROIs by compartment was performed. Results: Nearest neighbor analysis shows increasing median distance between tumor and immune cells from untreated, to NACT, and NACT+RNI, and no differences between short and standard pre-operative RNI. Proximity analysis reveals the distribution of CD3+ cells from the CK+ cells changes with increasing therapy, favoring frequency of closer immune cells, although the average number of cells around each CK+ cell diminishes with increasing therapies. CK+ cell density remains stable, suggesting migration rather than cell death. Differences in expression profiles of immune ROIs suggest more immune signaling in pre-op short. Considering all RT, 27 differentially expressed proteins (DEP) were significantly upregulated in Post tumor ROIs. None were significantly downregulated. After correction by LMM, 11 markers were up in Post cases and none downregulated. We found lower expression of SMA , CD4, CD45RO, CD14, CD11c, TIM-3, and B7-H3 in the Pre nodes. No significant DEPs remain in immune ROIs after LMM correction. The first two principal components accounted for 45.8% of the total variance, with PC1 largely separating samples based on Pre RNI. Pre-short vs Pre-standard showed 9 increased and 0 decreased proteins in Pre-short including CD27, Sting, CD40, CD11c, CD25. Conclusions: Pre-operative radiation contributes to Immune microenvironment remodeling, complementing neoadjuvant chemotherapy in ILN, without altering immune cell density. Different immune signals in irradiated ILN suggest a less immunosuppressive phenotype in RNI, and potential immune activation in Pre-short RNI. Small sample size limits conclusions, immune studies will follow to optimize radiation regimens. Citation Format: Stephanie O. Dudzinski, Elizve N. Barrientos-Toro, Benjamin D. Smith, Simona F. Shaitelman, Sharia Hernandez, Alejandra Serrano, Khan Khaja, Larisa Kostousov, Wei Lu, Rensi Zacharia, Nathan Comeaux, Solis M. Luisa, Aysegul A. Sahin, Maria Gabriela Raso, Jing Wang, Karen Hoffman, Wendy A. Woodward. Spatial protein profiling of irradiated ER positive breast cancer lymph nodes suggests nodal irradiation modulates immune function [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 7432.

Clinical and pathological characteristics of the 53 TNBC

This file contains all the information pertaining to supplementary methods that were referred to in the supplementary data.

This file includes all the 8 supplementary figures and their corresponding figure legends referred to in the main text.

Supplementary data, tables and legends

Immune cell subset annotations and antibodies used for analysis.

Stromal tumor-infiltrating lymphocytes (sTILs) may hold clinical value for triple-negative breast cancer (TNBC) for outcome prediction. Manual scoring of sTILs suffers from inter-observer variability, limiting its clinical adoption. We developed an artificial intelligence (AI) workflow to automatically quantify sTILs from diagnostic biopsies and evaluated its predictive performance for pathological complete response (pCR) in patients with early-stage TNBC receiving neoadjuvant chemotherapy. The pipeline combined tissue segmentation with context-aware cell classification to derive sTILs scoring, AI-sTILdensity. Using pretreatment biopsies (n = 394) from the ARTEMIS trial (ClinicalTrials.gov: NCT02276443; October 21, 2014), AI-sTILdensity showed strong correlation with manual sTILs (Spearman rho: 0.686-0.739) and improved prediction of pCR (AUC: 0.702-0.747 vs. 0.692-0.713) as an independent feature (P = 0.016). Predictive performance was consistent in an external TNBC set (n = 64). These findings demonstrated that automated assessment of sTILs from biopsy specimens is reproducible and can modestly enhance pCR prediction, supporting the use of AI approaches to enable more objective and scalable evaluation of sTILs in TNBC.

Human epidermal growth factor receptor 2 (HER2) is an established therapeutic target in multiple solid tumors, particularly breast and gastric cancers. Significant advancements have been made in the development of HER2-targeted therapies, including monoclonal antibodies, tyrosine kinase inhibitors, antibody–drug conjugates (ADC), and novel bispecific antibodies. These agents have revolutionized the treatment landscape for HER2-positive metastatic cancers, resulting in improved progression-free and overall survival, and quality of life for patients. Beyond breast and gastric cancers, HER2 expression/amplification has been observed in other solid tumors, such as colorectal, lung, bladder, ovarian, and biliary tract cancers, offering new opportunities for personalized therapy in a larger patient population. In this review, we highlight the current state of HER2-targeted treatment strategies across HER2-expressing solid tumors, discussing the clinical efficacy, adverse event profiles, and challenges of existing therapies. We explore emerging treatment approaches, including novel agents such as HER2-targeting ADCs, combination therapies, and strategies to overcome resistance mechanisms. Additionally, we examine the role of HER2 expression heterogeneity, biomarker-driven patient selection, and diagnostic tools for patient selection and optimization of treatment outcomes. This review also investigates ongoing challenges in expanding HER2-targeted therapies, including addressing intrinsic and acquired resistance and tailoring strategies to low HER2-expressing or HER2-mutant tumors. Lastly, we provide insights into future directions, emphasizing the importance of precision oncology to broaden the therapeutic opportunities of HER2-targeted therapies across diverse HER2-driven malignancies.

This file contains Supplementary Table 1, pertaining to the proteomic classification of RPPA antibodies

TROP2, a transmembrane glycoprotein implicated in tumor progression and metastasis, exhibits variable expression across breast cancer subtypes. The classic variant of invasive lobular carcinoma (ILC-C) and the pleomorphic variant (ILC-P) display distinct morphological and biological characteristics. This study aims to investigate differences in TROP2 expression between ILC-C and ILC-P, enhancing our understanding of its potential as a biomarker for aggressive subtypes. TROP2 protein expression was evaluated in 14 samples (8 ILC-C and 6 ILC-P) using immunohistochemistry (IHC) and quantified with digital image analysis software. Samples were annotated and classified into tumor and stroma components. The percentage of TROP2-positive cells was assessed, and staining intensity was categorized as weak, moderate, or strong. H-scores were calculated to assess overall TROP2 expression. Statistical analyses included descriptive statistics and the Wilcoxon test to compare expression levels between ILC-C and ILC-P groups. TROP2 expression was significantly higher in ILC-P samples compared to ILC-C across multiple metrics. The percentage of positive cells was markedly elevated in ILC-P (mean: 73.04; median: 81.09; SD: 16.08) compared to ILC-C (mean: 17.97; median: 15.14; SD: 12.48). Similarly, H-scores were significantly higher in ILC-P (mean: 153.07; median: 146.79; SD: 54.31) than in ILC-C (mean: 24.97; median: 17.96; SD: 18.81). The Wilcoxon test for both parameters revealed statistical significance (p = 0.002). However, no significant difference was observed between molecular classification groups and TROP2 expression levels. Our findings demonstrate distinct TROP2 protein expression profiles in ILC, with ILC-P samples exhibiting significantly higher expression levels. This elevated TROP2 expression in the aggressive ILC-P variant highlights its potential as a biomarker for identifying high-risk lobular carcinoma subtypes. Further studies are warranted to explore the clinical implications of TROP2 in ILC-P, potentially guiding targeted therapeutic strategies. Elizve Barrientos-Toro, Harsh Batra, Pandurengan Renganayaki, Yasmeen Qamar Rizvi, Jason Aboudi Mouabbi, Funda Funda Meric-Bersntam, Qingqing Ding, Aysegul Sahin, Maria Gabriela Raso. Differential TROP2 immunohistochemical expression between pleomorphic and classic invasive lobular carcinoma of the breast [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 4613.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. The epithelial-to-mesenchymal transition is a nonbinary process in the metastatic cascade that generates tumor cells with both epithelial and mesenchymal traits known as hybrid EM cells. Recent studies have elucidated the enhanced metastatic potential of cancers featuring the hybrid EM phenotype, highlighting the need to uncover molecular drivers and targetable vulnerabilities of the hybrid EM state. Here, we discovered that hybrid EM breast tumors are enriched in CD38, an immunosuppressive molecule associated with worse clinical outcomes in liquid malignancies. Altering CD38 expression in tumor cell impacted migratory, invasive, and metastatic capabilities of hybrid EM cells. Abrogation of CD38 expression stimulated an antitumor immune response, thereby preventing the generation of an immunosuppressive microenvironment in hybrid EM tumors. CD38 levels positively correlated with PD-L1 expression in samples from patients with TNBC. Moreover, targeting CD38 potentiated the activity of anti-PD-L1, eliciting strong antitumor immunity, with reduced tumor growth in hybrid EM models. Overall, this research exposes upregulation of CD38 as a specific survival strategy utilized by hybrid EM breast tumors to suppress immune cell activity and sustain metastasis, with strong implications in other carcinomas that have hybrid EM properties. Significance: Hybrid cells co-featuring epithelial and mesenchymal traits in triple-negative breast cancer express elevated levels of CD38 to induce immunosuppression and metastasis, indicating CD38 inhibition as potential strategy for treating breast cancer.

Breast carcinoma represents a biologically heterogeneous group of malignancies with diverse clinical behaviors, highlighting the need for robust molecular markers to support accurate diagnosis, prognosis, and therapeutic decision-making. Histopathologic evaluation and assessment of established biomarkers, including estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, remain central to clinical management, yet these biomarkers do not fully capture the disease complexity. Advances in genomics have enabled identification of intrinsic breast cancer subtypes, improving risk stratification and personalization of therapy. New molecular technologies such as next-generation sequencing and liquid biopsy approaches have uncovered actionable genomic alterations and allow for real-time monitoring. Emerging biomarkers such as cyclin E overexpression, BRCA1/2 mutations, and mismatch repair deficiency provide additional prognostic and predictive value, especially in guiding targeted therapies. The tumor microenvironment, including immune cell infiltration and stromal composition, has also gained recognition for its role in modulating tumor progression and therapeutic response. Liquid biopsy tools, including analysis of circulating tumor DNA, circulating tumor cells, and exosomes, offer promising non-invasive methods for disease surveillance and early detection of resistance. High-throughput techniques such as spatial transcriptomics and multiplex immunofluorescence are accelerating biomarker discovery with potential clinical application. Despite these advances, challenges remain in standardization, reproducibility, and clinical validation. This review summarizes current and emerging breast cancer biomarkers, emphasizing their role in advancing precision oncology. Continued translational research and methodological refinement are essential for successful integration of new biomarkers into clinical practice and for improving patient outcomes.

INTRODUCTION:Endocrine therapy combined with cyclin-dependent kinase 4 and 6 inhibitors (1) has been approved for patients with hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast carcinomas. It has been known that the p16-CDK4/6-cyclin D1 axis regulates the S phase of the cell cycle. We investigated the association between p16 expression and the therapeutic response with CDK4/6i in advanced breast carcinoma. METHODS:Patients diagnosed with invasive breast carcinoma between 2019 and 2024 whose tumors underwent next-generation sequencing (NGS) based analysis were identified. The expression of p16 was assessed in tumor cells and stromal cells within the invasive carcinoma by immunohistochemistry. In tumor cells, p16 expression was subclassified as cytoplasmic (TC), nuclear (TN), or cytoplasmic and nuclear staining (TCN). In stromal cells, p16 expression was assessed in tumor-associated fibroblasts (TAF) and tumor-infiltrating immune cells (TILs) in any cellular compartment. RESULTS:Among the 35 cases, p16-TC, -TN, and -TCN were significantly associated with disease progression during CDK4/6i therapy. The positive association between p16-TC and progressive disease remained in the 24 CDK4/6i-treatment naïve tumors. In contrast, the tumors with p16 expression in TAF showed a numerically higher response rate than the p16-TAF-negative ones. We also observed a significant association between p16-TC expression and lymph node metastasis. CONCLUSION:Our study demonstrated a significant association between p16 expression in tumor cells and an unfavorable therapeutic response to CDK4/6i in advanced breast carcinoma. The clinical significance of p16 expression patterns as a predictive biomarker for CDK4/6i deserves further investigation.

Fibroepithelial lesions (FELs) of the breast represent a diverse group of biphasic tumors with varying morphologies and clinical behavior. The classification of FELs is mainly based on a constellation of diagnostic criteria, and intralesional heterogeneity is not uncommon. Therefore, reporting FELs in a core needle biopsy (CNB) with limited tissue material can be challenging as not all the features may be represented for assessment. Differentiating a classic fibroadenoma from a well-sampled phyllodes tumor (PT) is generally straightforward. However, cellular fibroadenoma, morphologically heterogeneous benign PT, and myoid hamartoma can overlap histologically. Accurate grading of PT is also challenging on CNB and carries significant management implications. In this article, we provide an overview and propose a pragmatic approach to reporting FELs on CNB, particularly for lesions with overlapping features. Guidance using the UK/European "B" classification of FELs alongside descriptive reporting of the various lesions, is also presented to aid in management decisions.

Accurate diagnosis of metastatic breast carcinoma is essential for effective clinical management. Immunohistochemistry plays a pivotal role in confirming or excluding a breast origin. In this review, we discuss both traditional and newly identified breast cancer markers, with a particular focus on studies related to TRPS1, and share our experience in applying this marker. Additionally, we explore the challenges associated with using these markers to differentiate metastatic breast cancer from malignancies of other origins.

Phyllodes tumours (PTs) of the breast present diagnostic challenges due to their complex histological features and potential for malignant behaviour. The World Health Organisation (WHO) classification requires the presence of five adverse histological criteria to categorise PTs as malignant, aiming to avoid overdiagnosis and improve diagnostic consistency. However, emerging evidence suggests that these strict criteria may underdiagnose tumours with metastatic potential and histological features that would otherwise be considered malignant in soft tissue tumours, leading to significant implications for prognosis and treatment. Recent studies have highlighted cases where tumours classified as borderline PT by WHO criteria exhibited metastatic behaviour, emphasising the need to refine the diagnostic framework. Microscopic criteria used to classify PT also vary among reporting pathologists, resulting in suboptimal reproducibility. This review examines the histological parameters utilised in the classification of malignant PT, highlights existing evidence gaps and analyses international breast pathologist survey data to propose a pragmatic diagnostic approach. We recommend redefining malignant PTs to include cases meeting four of the five WHO criteria, supplemented by comprehensive sampling and clinical context. This approach balances the risk of underdiagnosis with the need for standardised, reproducible diagnostic practices. Future collaborative efforts should focus upon developing evidence-based, biologically relevant classification systems and leveraging technological advancements to enhance diagnostic precision. These efforts aim to refine classification, improve prognostic accuracy and optimise patient management strategies.

This file contains all the supplementary materials and methods referred to in the text.

PURPOSE:Trastuzumab deruxtecan (T-DXd) is currently approved for treating metastatic breast cancer (MBC) that is HER2 positive [immunohistochemistry (IHC) score of 3+ or in situ hybridization (ISH) positivity] or HER2-low (IHC score of 1+ or IHC 2+/ISH negative), as well as for HER2-positive gastric cancer, HER2-mutant lung cancer, and HER2-overexpressing solid tumors. Given the increasing utilization of T-DXd, we sought to determine how HER2 status might change following T-DXd therapy. EXPERIMENTAL DESIGN:We retrospectively reviewed patients with MBC who received T-DXd at the University of Texas MD Anderson Cancer Center. We included patients with paired pre- and post-treatment biopsies assessed for HER2 status using IHC. RESULTS:We included 41 patients with MBC who received treatment with T-DXd and had paired pre- and post-treatment biopsies assessed for HER2 status using IHC. HER2 loss was observed in 11 patients [32.4% of 34 patients with pre-treatment HER2 expression (1+, 2+, or 3+)] following treatment with T-DXd. In addition to the 11 patients with HER2 loss, another 10 patients (29.4%) had a decrease in HER2 score after treatment with T-DXd. CONCLUSIONS:HER2 loss and decrease in HER2 expression are common in patients with MBC receiving treatment with T-DXd. Reevaluation of HER2 status following T-DXd treatment should be considered prior to alternate HER2-targeted therapies that require HER2 overexpression for efficacy.

Postradiation angiosarcoma of the breast is a secondary angiosarcoma in breast carcinoma patients who have undergone radiation therapy. Although postradiation angiosarcoma is rare, it is very aggressive and has an extremely poor prognosis. Previous studies have implicated matrix metalloproteinase 9 (MMP9) in some cancer development and progression and angiogenesis. In this study, we compared MMP9 expression in surgical specimens from breast cancer patients treated with radiation who did and did not develop breast angiosarcoma within 15 years. We also compared MMP expression in the breast angiosarcoma specimens from patients with postradiation angiosarcoma of the breast and patients with non-breast angiosarcoma not previously treated with radiation. Our results showed that MMP9 was expressed in the endothelial cells in the breast carcinoma specimens from patients who developed angiosarcoma after radiation therapy but not in the breast carcinoma specimens from patients without angiosarcoma. In addition, MMP9 expression was higher in patients with postradiation angiosarcoma of the breast than in patients with non-radiation-associated primary angiosarcomas at non-breast sites. These results suggest that MMP9 may be a risk factor for and play a significant role in the development of postradiation angiosarcoma of the breast.