Early-stage Invasive Breast Cancer Research Articles

Tripartite Motif-Containing 2, a Glutamine Metabolism-Associated Protein, Predicts Poor Patient Outcome in Triple-Negative Breast Cancer Treated with Chemotherapy

Background: Breast cancer (BC) remains heterogeneous in terms of prognosis and response to treatment. Metabolic reprogramming is a critical part of oncogenesis and a potential therapeutic target. Glutaminase (GLS), which generates glutamate from glutamine, plays a role in triple-negative breast cancer (TNBC). However, targeting GLS directly may be difficult, as it is essential for normal cell function. This study aimed to determine potential targets in BC associated with glutamine metabolism and evaluate their prognostic value in BC. Methods: The iNET model was used to identify genes in BC that are associated with GLS using RNA-sequencing data. The prognostic significance of tripartite motif-containing 2 (TRIM2) mRNA was assessed in BC transcriptomic data (n = 16,575), and TRIM2 protein expression was evaluated using immunohistochemistry (n = 749) in patients with early-stage invasive breast cancer with long-term follow-up. The associations between TRIM2 expression and clinicopathological features and patient outcomes were evaluated. Results: Pathway analysis identified TRIM2 expression as an important gene co-expressed with high GLS expression in BC. High TRIM2 mRNA and TRIM2 protein expression were associated with TNBC (p < 0.01). TRIM2 was a predictor of poor distant metastasis-free survival (DMFS) in TNBC (p < 0.01), and this was independent of established prognostic factors (p < 0.05), particularly in those who received chemotherapy (p < 0.05). In addition, TRIM2 was a predictor of shorter DMFS in TNBC treated with chemotherapy (p < 0.01). Conclusions: This study provides evidence of an association between TRIM2 and poor patient outcomes in TNBC, especially those treated with chemotherapy. The molecular mechanisms and functional behaviour of TRIM2 and the functional link with GLS in BC warrant further exploration using in vitro models.

Clinicopathological Significance of Cyclin-Dependent Kinase 2 (CDK2) in Ductal Carcinoma In Situ and Early-Stage Invasive Breast Cancers.

Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.

Abstract PO2-07-09: Early stage invasive lobular breast cancer is underestimated on conventional imaging including MRI

Abstract Background: Invasive lobular carcinoma (ILC), representing 15% of all invasive breast cancers, is characterized by loss or dysfunction of the adhesion molecule E-cadherin. This leads to an infiltrative growth pattern that is unlikely to disrupt the normal architecture of breast tissue. As a result, ILC is often non-mass forming and imaging is limited in correctly diagnosing stage I-III ILC. Mammography is believed to underestimate ILC, while MRI tends to overestimate the extent of ILC. The aim of this retrospective study was to correlate radiological with pathological findings. Methods: All patients diagnosed with stage I-III pure (i.e. not mixed) ILC between January 2000 and December 2020 who underwent primary surgery in University Hospitals Leuven, were included. Data on patient characteristics, preoperative imaging and pathology were collected from the patient files. Imaging and pathology measurements were compared by use of Pearson correlation coefficient, Whitney U test or Chi-square test. Weighted kappa statistics were used to assess agreement. Since the techniques of mammography and ultrasound have evolved in the past 20 years, different time periods were considered in sub-analyses (2000-2004, 2005-2009, 2010-2014, 2015-2020). Results: In total 1029 patients were included. Median age at diagnosis was 61.0 years (range 32.0 – 95.0 years). The breast density score determined on mammogram was Bi-RADS type A for 52 (5.1%) patients, B for 238 (23.1%) patients, C for 235 (22.8%) patients and D for 141 (13.7%) patients. Density score was missing from the reports of the remaining patients (n=363, 35.3%). Contrast enhanced breast magnetic resonance imaging (MRI) was performed in 709 (68.9%) patients. An increase in preoperative use of MRI was seen over the years. In comparison to tumor size reported by pathology, all imaging techniques underestimated the tumor size. The mean difference in largest tumor size compared to pathology was 14.64 ± 21.15 mm for mammography, 18.19 ± 21.66 mm for ultrasound and 9.93 ± 20.63 mm for MRI. A higher breast density level and a larger tumor size were significantly associated with a larger difference between diameter on pathology versus mammography (ρ=0.102, p-value 0.025 and ρ=0.530, p-value &amp;lt; 0.001 respectively). Changes over different time periods are shown in Table 1 for mammography and ultrasound. There was an agreement on unifocality versus multifocality between pathology and mammography for 81.2% of the patients. In 52.1% of the 236 cases where multifocality was reported on MRI, only 1 lesion was reported by the pathologist. Multifocality was seen on pathology in 12.0% of the 460 cases that were seen as unifocal lesions on MRI. Considering adenopathies, the false positive rate of ultrasound was 3.0%. However, the false negative rate was 68.3%. Conclusions: The local extent of ILC is underestimated by conventional imaging techniques. Unlike previous reports, our results suggest that tumor size of ILC is also underestimated by MRI. Ultrasound was inferior to mammography and MRI in estimating tumor size in our series. It was confirmed that MRI tends to overestimate the number of foci which might lead to unnecessary secondary ultrasounds and biopsies. The presurgical underestimation of lymph node involvement might increase the need of secondary surgeries. It is crucial to address these limitations in imaging of ILC and to prioritize the development of enhanced imaging techniques to improve diagnostic accuracy for these patients. Table 1: difference between imaging techniques and pathology by year of diagnosis Citation Format: Kaat Van Herck, Karen Van Baelen, Chantal Van Ongeval, Valerie Celis, Helen De Boodt, Machteld Keupers, Renate Prevos, Giuseppe Floris, Ines Nevelsteen, Ann Smeets, Thaïs Baert, Sileny Han, Hans Wildiers, Annouschka Laenen, Christine Desmedt, Patrick Neven. Early stage invasive lobular breast cancer is underestimated on conventional imaging including MRI [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 PO2-07-09.

Abstract PO1-07-01: Artificial intelligence-based prediction of Oncotype DX Score from whole slide images using human-interpretable features and breast biomarkers

Abstract Background The Oncotype DX Breast Recurrence Score assay (ODX) is a commonly used genomic test for patients with estrogen receptor (ER)-positive, HER2-negative, early-stage invasive breast cancer. While ODX predicts patients’ recurrence risk and benefit from chemotherapy, it is tissue and time-consuming, and expensive. Previous deep-learning or non-linear ODX prediction models achieved promising performance using whole-slide images (WSI) or with other covariates (e.g., ER, progesterone receptor (PR), HER-2, Ki-67 scores and tumor stage) but systematic quantification of the contribution of individual histological features to the ODX score remained challenging. Here, we extracted a rich set of human-interpretable features (HIFs) quantifying nuclear morphology and the distribution of cells and tissues in the tumor microenvironment. We used these HIFs, along with manually assessed ER, PR, HER-2 scores, and tumor stage, to predict ODX scores. We also explored the role of Ki-67 features in augmenting our model predictions Methods We developed machine learning models to extract cell, tissue, and nuclear HIFs from WSI stained with hematoxylin and eosin (H&amp;E) and immunohistochemistry (IHC) against Ki-67. These models were deployed on 353 H&amp;E WSI to extract 276 HIFs quantifying cell densities, tissue areas, relative cell counts, and nuclei size, shape and color. Hierarchical agglomerative clustering was applied to cluster these features into 11 groups. Univariate regression was performed to identify correlations between ODX scores and feature clusters. To predict ODX score using HIFs, a multivariable regression model was fitted to the data where regressors are representative features from each cluster, in addition to ER, PR, HER-2 scores and stage as covariates. The model was fitted on the training set (N=266 slides) and evaluated on the held-out test set (N=87 slides). To examine the utility of Ki-67 IHC features in ODX prediction, a Ki-67 model was deployed on a subset of 194 matched Ki-67 IHC WSI to extract count proportions and densities of Ki-67 positive cells in cancer epithelium and stroma. Results Significant positive correlations were identified between ODX scores and feature clusters corresponding to cancer cell density (p &amp;lt; 10-7), macrophage density (p &amp;lt; 10-4), immune cell density (p &amp;lt; 10-16), and variations in cancer nuclear size (p &amp;lt; 10-5) and color (p &amp;lt; 10-3). Significant negative correlations were observed between ODX scores and clusters related to fibroblast density (p &amp;lt; 10-3), and variations of non-cancer cell nuclear color (p = 0.02). Evaluation of our model’s ability to predict ODX scores revealed an association between predicted and observed scores (Pearson r = 0.74). The AUROC for model predictions of high/low classifications (with reference to a cutoff ODX score of 20) was 0.80. Ki-67 features were clustered together with H&amp;E cancer cell density features and showed a stronger correlation with ODX scores compared to the H&amp;E features alone. Combining Ki-67 cell density features with H&amp;E features led to increased performance of the multivariable regression model on the test set compared to H&amp;E features only (r = 0.62 vs. r = 0.59). However, a model combining Ki-67, H&amp;E and covariates performed similarly to a model with H&amp;E and covariates (r = 0.73), suggesting that Ki-67 features may not be needed for ODX prediction when H&amp;E and covariate information is available. Conclusions Our model predicts ODX recurrence scores with comparable performance to other black-box approaches using WSI and breast biomarker status and without the need for, Ki-67 features. Using readily available information, our model has the potential to be a convenient screening tool for patient stratification, which may lead to better patient care, lower costs, and faster treatment. Citation Format: Nhat Le, John Van Arnam, Christian Kirkup, Ylaine Gerardin, Michael Drage, Neha Khaitan, Anurag Sharma, Georgi Galev, Nicholas Indorf, Emily Sansevere, Ilan Wapinski, Stephanie Hennek, Gloria Zhang. Artificial intelligence-based prediction of Oncotype DX Score from whole slide images using human-interpretable features and breast biomarkers [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 PO1-07-01.

Abstract PO5-15-11: A Novel Biosignature to Predict Radiation Therapy Response in Early-Stage Invasive Breast Cancer Treated with Breast-Conserving Surgery

Abstract Introduction: Radiation therapy (RT) is a standard component of treatment for most patients with early-stage, hormone receptor-positive, invasive breast cancer (BC) following breast-conserving surgery (BCS). RT recommendations are primarily based on clinicopathologic (CP) factors, such as tumor size, nodal status, and histological grade. While these factors provide prognostic information, their ability to accurately identify patients at a higher risk of ipsilateral breast recurrence (IBR) or those who will significantly benefit from RT is unclear. Therefore, incorporation of additional predictive markers beyond CP factors is warranted and there remains a need to refine the selection criteria for patients who will derive the greatest benefit from RT. In this study, we aimed to explore the role of immune and metabolic signaling axes as potential predictive markers for RT response in hormone receptor-positive breast cancer patients. Methods: A cohort of 939 women from Sweden were diagnosed with BC between 1987 and 2004. A subset of 471 patients was diagnosed with T1-T2N0M0 BC and underwent BCS with negative margins. Patients were treated with BCS plus/minus RT (median dose 50Gy) based upon physician preference. Biomarkers were assayed in formalin-fixed paraffin-embedded tissue micro arrays using multiplex immunofluorescence and multi-spectral imaging in a CLIA lab (Laguna Hills, CA). Immune and metabolic axes were defined employing biomarkers and used to classify patients into three risk groups: 1) Low Risk (lower baseline risk and no RT benefit), 2) Elevated Risk (higher baseline risk and significant RT benefit), and 3) Residual Risk (higher baseline risk and suboptimal RT benefit). Risk groups were analyzed for IBR rate using Kaplan-Meier and Cox proportional hazards analyses. Risk group prognosis and RT prediction was assessed for RT-risk group interaction in multivariable analysis, adjusting for CP factors. Results: Among the patients with complete biomarker data, 261 were hormone receptor-positive, and had a median follow-up of 140 months. Median age was 62 years (28-88 years), and median tumor size was 1.1 cm (0.1-4 cm). Some patients (21%) received endocrine therapy (ET), and most (76%) received RT, with 79% of patients who received ET also receiving RT. In the overall population, RT following BCS was associated with a reduction in HR for recurrence (HR=0.3, 95%CI [0.1, 0.5], p&amp;lt; 0.001). Patients classified as Low Risk (41%) did not have a significant benefit from RT (HR=0.9, 95%CI [0.1, 8.3], p=0.95) in multivariable analysis adjusted for CP factors. For patients who were classified as Elevated Risk (31%), multivariable analysis showed a higher IBR rate without RT (HR=5.7, 95%CI [1.3, 25.1], p&amp;lt; 0.021) relative to the Low Risk group who had a significant benefit from RT (HR=0.1, 95%CI [0.02, 0.2], p&amp;lt; 0.001). Patients classified into the Residual Risk group (28%) had no significant reduction from RT (HR=0.7, 95%CI [0.2, 2.0], p=0.61). However, in multivariable analysis adjusted for CP factors, patients treated with RT in the Residual Risk group had higher IBR rates (HR=3.7, 95%CI [1.6, 8.7], p=0.0026) compared to RT-treated patients in other risk groups. The biosignature was predictive for RT benefit in multivariable analysis adjusted for CP factors (p-interaction &amp;lt; 0.001). Conclusions:The biosignature used in this study demonstrated the potential to identify patients who may derive limited benefit from RT, allowing for tailored therapeutic approaches. These findings underscore the importance of individualized treatment decisions based on risk stratification. Further clinical validation and refinement of risk stratification strategies are warranted to optimize outcomes in hormone receptor-positive, BC patients undergoing BCS. Citation Format: Troy Bremer, Karuna Mittal, Chirag Shah, Frank Vicini, Steven Shivers, Charlotta Wadsten. A Novel Biosignature to Predict Radiation Therapy Response in Early-Stage Invasive Breast Cancer Treated with Breast-Conserving Surgery [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 PO5-15-11.

Increase in Hypofractionated Radiation Therapy Among Patients with Invasive Breast Cancer or Ductal Carcinoma In Situ: Who is Left Behind?

We aimed to update the trend of hypofractionated whole-breast irradiation (HF-WBI) use over time in the US and examine factors associated with lack of HF-WBI adoption for patients with early-stage invasive breast cancer (IBC) or ductal carcinoma in situ (DCIS) undergoing a lumpectomy. Among patients who underwent a lumpectomy, we identified 928,034 early-stage IBC patients and 330,964 DCIS patients in the 2004-2020 National Cancer Database. We defined HF-WBI as 2.5-3.33 Gy/fraction to the breast and conventional fractionated whole-breast irradiation as 1.8-2.0 Gy/fraction. We evaluated the trend of HF-WBI utilization using a generalized linear model with the log link and binomial distribution. Factors associated with HF-WBI utilization were assessed using multivariable logistic regression in patients diagnosed between 2018 and 2020. Among IBC patients, HF-WBI use has significantly increased from 0.7% in 2004 to 63.9% in 2020. Similarly, HF-WBI usage among DCIS patients has also increased significantly from 0.4% in 2004 to 56.6% in 2020. Black IBC patients were less likely than White patients to receive HF-WBI (adjusted odds ratio [AOR] 0.81, 95% CI: 0.77-0.85). Community cancer programs were less likely to administer HF-WBI to IBC patients (AOR 0.80, 95% CI: 0.77-0.84) and to those with DCIS (AOR 0.87, 95% CI: 0.79-0.96) than academic/research programs. Younger age, positive nodes, larger tumor size, low volume programs, and facility location were also associated with lack of HF-WBI adoption in both patient cohorts. HF-WBI utilization among post-lumpectomy patients has significantly increased from 2004-2020 and can finally be considered standard of care in the US. We found substantial disparities in adoption within patient and facility subgroups. Reducing disparities in HF-WBI adoption has the potential to further alleviate healthcare costs while improving patients' quality of life.

Axillary ultrasonography for early-stage invasive breast cancer

BackgroundAmong women with early invasive breast cancer and 1–2 positive sentinel nodes, sentinel lymph node biopsy (SLNB) is non-inferior to axillary lymph node dissection (ALND).1-3 However, preoperative axillary ultrasonography (AxUS) may not be sensitive enough to discriminate burden of nodal metastasis in these patients, potentially leading to overtreatment.4-6 This study compares axillary operation rates in patients who did and did not receive preoperative AxUS, assessing its utility and risks for overtreatment. MethodsThis is a retrospective cohort study of patients with clinical T1/T2 breast tumors who were clinically node negative and underwent an axillary operation. ResultsPatients who had preoperative AxUS received more ALND compared to patients who did not (5.6% vs. 1.4%, p ​< ​0.001). There was no significant difference in the number of additional axillary operations following SLNB (2.1% vs. 2.3%, p ​= ​0.77). ConclusionEliminating preoperative AxUS is associated with fewer invasive ALND procedures, without increased rate of axillary reoperations.

Identification of MicroRNAs Associated with Histological Grade in Early-Stage Invasive Breast Cancer.

This study aimed to identify microRNAs associated with histological grade using comprehensive microRNA analysis data obtained by next-generation sequencing from early-stage invasive breast cancer. RNA-seq data from normal breast and breast cancer samples were compared to identify candidate microRNAs with differential expression using bioinformatics. A total of 108 microRNAs were significantly differentially expressed in normal breast and breast cancer tissues. Using clinicopathological information and microRNA sequencing data of 430 patients with breast cancer from The Cancer Genome Atlas (TCGA), the differences in candidate microRNAs between low- and high-grade tumors were identified. Comparing the expression of the 108 microRNAs between low- and high-grade cases, 25 and 18 microRNAs were significantly upregulated and downregulated, respectively, in high-grade cases. Clustering analysis of the TCGA cohort using these 43 microRNAs identified two groups strongly predictive of histological grade. miR-3677 is a microRNA upregulated in high-grade breast cancer. The outcome analysis revealed that patients with high miR-3677 expression had significantly worse prognosis than those with low miR-3677 expression. This study shows that microRNAs are associated with histological grade in early-stage invasive breast cancer. These findings contribute to the elucidation of a new mechanism of breast cancer growth regulated by specific microRNAs.

Non-invasive prediction model of axillary lymph node status in patients with early-stage breast cancer: a feasibility study based on dynamic contrast-enhanced-MRI radiomics.

Accurate axillary evaluation plays an important role in prognosis and treatment planning for breast cancer. This study aimed to develop and validate a dynamic contrast-enhanced (DCE)-MRI-based radiomics model for preoperative evaluation of axillary lymph node (ALN) status in early-stage breast cancer. A total of 410 patients with pathologically confirmed early-stage invasive breast cancer (training cohort, N = 286; validation cohort, N = 124) from June 2018 to August 2022 were retrospectively recruited. Radiomics features were derived from the second phase of DCE-MRI images for each patient. ALN status-related features were obtained, and a radiomics signature was constructed using SelectKBest and least absolute shrinkage and selection operator regression. Logistic regression was applied to build a combined model and corresponding nomogram incorporating the radiomics score (Rad-score) with clinical predictors. The predictive performance of the nomogram was evaluated using receiver operator characteristic (ROC) curve analysis and calibration curves. Fourteen radiomic features were selected to construct the radiomics signature. The Rad-score, MRI-reported ALN status, BI-RADS category, and tumour size were independent predictors of ALN status and were incorporated into the combined model. The nomogram showed good calibration and favourable performance for discriminating metastatic ALNs (N + (≥1)) from non-metastatic ALNs (N0) and metastatic ALNs with heavy burden (N + (≥3)) from low burden (N + (1-2)), with the area under the ROC curve values of 0.877 and 0.879 in the training cohort and 0.859 and 0.881 in the validation cohort, respectively. The DCE-MRI-based radiomics nomogram could serve as a potential non-invasive technique for accurate preoperative evaluation of ALN burden, thereby assisting physicians in the personalized axillary treatment for early-stage breast cancer patients. This study developed a potential surrogate of preoperative accurate evaluation of ALN status, which is non-invasive and easy-to-use.

Publisher's Note

Publisher's Note

Machine Learning-Based Breast Tumor Ultrasound Radiomics for Pre-operative Prediction of Axillary Sentinel Lymph Node Metastasis Burden in Early-Stage Invasive Breast Cancer

The aim of the work described here was to assess the application of ultrasound (US) radiomics with machine learning (ML) classifiers to the prediction of axillary sentinel lymph node metastasis (SLNM) burden in early-stage invasive breast cancer (IBC). In this study, 278 early-stage IBC patients with at least one SLNM (195 in the training set and 83 in the test set) were studied at our institution. Pathologic SLNM burden was used as the reference standard. The US radiomics features of breast tumors were extracted by using 3D-Slicer and PyRadiomics software. Four ML classifiers-linear discriminant analysis (LDA), support vector machine (SVM), random forest (RF) and decision tree (DT)-were used to construct radiomics models for the prediction of SLNM burden. The combined clinicopathologic-radiomics models were also assessed with respect to sensitivity, specificity, accuracy and areas under the curve (AUCs). Among the US radiomics models, the SVM classifier achieved better predictive performance with an AUC of 0.920 compared with RF (AUC = 0.874), LDA (AUC = 0.835) and DT (AUC = 0.800) in the test set. The clinicopathologic model had low efficacy, with AUCs of 0.678 and 0.710 in the training and test sets, respectively. The combined clinicopathologic (C) factors and SVM classifier (C + SVM) model improved the predictive ability with an AUC of 0.934, sensitivity of 86.7%, specificity of 89.9% and accuracy of 91.0% in the test set. ML-based US radiomics analysis, as a novel and promising predictive tool, is conducive to a precise clinical treatment strategy.

Analytical Validation of the PreciseDx Digital Prognostic Breast Cancer Test in Early-Stage Breast Cancer

BackgroundPreciseDx Breast (PDxBr) is a digital test that predicts early-stage breast cancer recurrence within 6-years of diagnosis. Materials and MethodsUsing hematoxylin and eosin-stained whole slide images of invasive breast cancer (IBC) and artificial intelligence-enabled morphology feature array, microanatomic features are generated. Morphometric attributes in combination with patient's age, tumor size, stage, and lymph node status predict disease free survival using a proprietary algorithm. Here, analytical validation of the automated annotation process and extracted histologic digital features of the PDxBr test, including impact of methodologic variability on the composite risk score is presented. Studies of precision, repeatability, reproducibility and interference were performed on morphology feature array-derived features. The final risk score was assessed over 20-days with 2-operators, 2-runs/day, and 2-replicates across 8-patients, allowing for calculation of within-run repeatability, between-run and within-laboratory reproducibility. ResultsAnalytical validation of features derived from whole slide images demonstrated a high degree of precision for tumor segmentation (0.98, 0.98), lymphocyte detection (0.91, 0.93), and mitotic figures (0.85, 0.84). Correlation of variation of the assay risk score for both reproducibility and repeatability were less than 2%, and interference from variation in hematoxylin and eosin staining or tumor thickness was not observed demonstrating assay robustness across standard histopathology preparations. ConclusionIn summary, the analytical validation of the digital IBC risk assessment test demonstrated a strong performance across all features in the model and complimented the clinical validation of the assay previously shown to accurately predict recurrence within 6-years in early-stage invasive breast cancer patients.

Deep (phospho)proteomics profiling of pre- treatment needle biopsies identifies signatures of treatment resistance in HER2+ breast cancer

Patients with early-stage HER2-overexpressing breast cancer struggle with treatment resistance in 20%-40% of cases. More information is needed to predict HER2 therapy response and resistance invivo. In this study, we perform (phospho)proteomics analysis of pre-treatment HER2+ needle biopsies of early-stage invasive breast cancer to identify molecular signatures predictive of treatment response to trastuzumab, pertuzumab, and chemotherapy. Our data show that accurate quantification of the estrogen receptor (ER) and HER2 biomarkers, combined with the assessment of associated biological features, has the potential to enable better treatment outcome prediction. In addition, we identify cellular mechanisms that potentially precondition tumors to resist therapy. We find proteins with expression changes that correlate with resistance and constitute to a strong predictive signature for treatment success in our patient cohort. Our results highlight the multifactorial nature of drug resistance invivo and demonstrate the necessity of deep tumor profiling.

A Biosignature Integrating Immune and Metabolic Signaling Axes to Assess Limited Radiation Therapy Response in Early-Stage Breast Cancer from a Low-Risk Cohort

Radiation therapy (RT) post breast conserving surgery (BCS) reduces in-breast recurrence (IBR) rate in early-stage invasive breast cancer (BC) patients. The RT treatment recommendation is often driven by clinicopathological (CP) factors; however, CP factors alone have limited ability to identify which women significantly benefit from RT, or those with higher IBR risk after BCS plus RT. Biologic factors driving unique phenotypes, in addition to CP, may improve prediction of RT response. In this study we evaluated the role of immune and metabolic signaling axes in predicting RT response in hormone receptor positive, HER2 negative, early-stage BC patients. Biomarkers from immune and metabolic signaling axes were studied in a cohort of 939 women from Sweden, at a CLIA certified lab (Laguna Hills, CA). Formalin fixed paraffin embedded tissues were assayed for protein expression using multiplex immunofluorescence and multi-spectral imaging. Immune and metabolic axes were assessed using biomarkers combined with a non-linear model, adjusting for patient age. RT prediction by the model was assessed, along and adjusted for CP factors and also among patients over 50-yrs. The model defined patient risk groups that were analyzed for IBR rate using Kaplan Meier analyses and Cox proportional hazards to test for RT-risk group interaction. Within the cohort, 440 patients had hormone receptor positive, HER2 negative BC treated with BCS (negative margins) and +/- RT without chemotherapy, where 296 patients had complete biomarker data. CP factors individually were not predictive for RT benefit, but grade was prognostic for IBR rate (p = 0.02) after BCS without RT. In multivariable analysis, adjusting for CP factors (grade, palpability, continuous size and age), the model was predictive for RT benefit (p-interaction = 0.046), identifying patients (n = 129) with worse RT benefit (HR = 7.8) compared to baseline RT benefit. The model was not prognostic for IBR rate in patients treated with BCS without RT (16% 10-yr IBR rate) but identified patients with increased IBR rates after BCS plus RT (HR = 3.9, p<0.001), where corresponding 10-yr IBR rates increased from 3% to 15%. The model was also predictive for RT benefit in women over 50-yrs (p-interaction = 0.05). The model identified 28% of women over 50-yrs who had increased IBR rates after BCS plus RT (HR = 4.0, p = 0.004), where corresponding 10-yr IBR rates increased from 3% to 12%. The model incorporating metabolic and immune signaling axes assessed in the study was predictive for RT benefit among women with early-stage hormone receptor positive, HER2 negative BC. While CP factors were not predictive of RT benefit, the inclusion of metabolic and immune signaling axes improved identification of patients with high residual risk after BCS plus RT and can potentially aid in personalized treatment of early-stage breast cancer based on individualized risk.

Percutaneous microwave ablation combined with endocrine therapy versus standard therapy for elderly patients with HR-positive and HER2-negative invasive breast cancer: a propensity score-matched analysis of a multi-center, prospective cohort study.

There is a growing trend to apply minimally invasive local treatments for elderly patients with early-stage breast cancer. As a minimally invasive thermal therapy, microwave ablation (MWA) has been attempted to treat breast cancer of small lesions, but its long-term local efficacy on elderly patients has seldom been reported. In this study, we aimed to compare outcomes of MWA combined with endocrine therapy to standard surgery combined with adjuvant therapy in the treatment of hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer for elderly patients. This prospective multi-center cohort study enrolled patients over 70 years old diagnosed with HR-positive and HER2-negative early-stage invasive breast cancer between January 2016 and July 2021. Patients chose either non-randomized to undergo MWA combined with endocrine therapy (MWA group) or standard surgery combined with adjuvant therapy (surgery group). Endpoints for the comparisons were disease-free survival (DFS), overall survival (OS) and length of hospital stay (LOS) after adjusting for previously reported risk factors using propensity score matching (1:3). Of the enrolled 132 patients, 33 were in the MWA group and 99 were in the surgery group. MWA was successfully performed in all cases, and technical effectiveness was achieved in all cases. With a median follow-up of 31 months, only one case had local recurrence 23 months after MWA. MWA combined with endocrine therapy and standard surgery combined adjuvant therapy for elderly patients with breast cancer achieved similar DFS [hazard ratio, 0.536; 95% confidence interval (CI): 0.128-2.249] and OS (hazard ratio, 0.537; 95% CI: 0.089-3.235). Besides, MWA had much shorter LOS than standard surgery (7.1 versus 13.0 days, P<0.001). MWA combined with endocrine therapy and standard surgery combined with adjuvant therapy for elderly breast cancer patients achieved similar outcomes. MWA combined with endocrine may be a feasible treatment strategy for elderly patients with HR-positive and HER2-negative invasive breast cancer.

EE269 Cost-Effectiveness of Profile Omission of Local Adjuvant Radiotherapy (POLAR), A Novel Genomic Signature, after Breast Conserving Surgery for Women with Invasive Early-Stage Breast Cancer (ESBC)

Adjuvant radiotherapy (RT), the standard of care for women diagnosed with invasive ESBC after breast-conserving surgery (BCS), is shown to reduce the risk of recurrence for most women; however, it can be associated with major adverse effects including lung/cardiac/breast toxicity. Recently, the POLAR signature was reported to successfully identify women who would or would not benefit from RT after BCS. This study evaluated the cost-effectiveness of the POLAR signature for guiding RT omission compared to RT for all women with hormone receptor-positive (HR+), HER2-negative stage I/IIA breast cancer.

Clinical Pathology of Breast Cancer: Staging and Biomarkers

Based on recent statistics, one in every eight women will be diagnosed with breast cancer in their life. Breast cancer is classified into a staging system that ranges from zero to four, which is determined by several factors. In the staging system, the clinical pathology taken and tested by the healthcare professional plays a key role in determining the gravity of the patient's case. Biomarkers provide insight into the carcinogenic cells in the person's body, such as the Estrogen receptor (ER), human epidermal growth factor (HER2), and the progesterone receptor which are discussed in this paper. Each of these biomarkers communicates a specific attribute about the tumor cells and signifies the severity and the extent of the breast cancer. Based on this research, it was proven that there is not a direct correlation, but a slight correlation between the biomarkers found in the cells with the relative stage the patient has. Studies have shown a link between cells testing HER2 positive with high-grade tumors, unfavorable prognosis, lymph node collusion, high mortality rates, and higher rates of recurrence in patients which signify a stage three or four cancer. Studies also suggested that patients who test ER positive, PR negative, and HER2 negative are more likely to have early-stage invasive breast cancer, which alludes to stage one or two clinically. A definite conclusion was not able to be obtained as more research must be conducted, however, general trends were discovered between stages and biomarkers.

166P A mono-institutional experience of field-in-field 3D-CRT planning in breast radiotherapy with FAST and FAST-forward protocols

The benefits of hypofractionated radiotherapy with FAST and FAST-forward protocols could be compromised by soft tissue reactions and cardiac toxicity caused by dose inhomogeneity and inaccurate heart sparing. This study aims to evaluate the dosimetric results of whole-breast hypofractionated radiotherapy with the field-in-field 3D conformal radiotherapy (3D-CRT) technique. A mono-institutional consecutive cohort of 122 early-stage invasive breast cancer patients was treated using field-in-field 3D-CRT. Thirty-nine patients were treated with 28.5 Gy in 5 fractions once a week (FAST protocol) and eighty-three patients with 26 Gy in 5 consecutive fractions (FAST-forward protocol). Different parameters were evaluated to assess target coverage, dose conformity and homogeneity and hot spots in unspecified tissues. To assess breast dimensions, nipple-to-pectoral muscle distance (NPD) and maximum mediolateral thickness (MLT) along tangential fields were measured. Evaluated OARs were ipsilateral lung and heart with their respective dose constraints as required by the clinical protocols. The median NPD and MLT were 4.65 cm [1.70-9.30] and 21.55 cm [11.26-32.30] respectively, with a median CTV of 380.50 cm3 [60.29-1255.13]. The median V95% was 99.45% [95.19-100], and the median 105% isodose volume was 0.74 cm3 [0.00-48.02] of whom 0.07 cm3 [0.00-5.22] in the first skin centimetre. This led to a median CI of 0.53 [0.33-1.00] and a median HI of 0.07 [0.03-0.19]. OARs dose-volume constraints were always respected. The median ipsilateral lung V8Gy was 7.32% [0.31-25.15]; as for the heart (left breast only), the median V1.5Gy was 6.45% [0.00-25.12] and median V7Gy was 0.34% [0.00-4.50]. Each plan achieved a clinically acceptable target coverage and homogeneity, with reduced superficial hot spots. These results suggested that field-in-field 3D-CRT can provide good quality FAST and FAST-forward breast RT plans, with the advantages of an efficient and cost-effective delivery technique. The clinical follow-up will give further critical feedback on treatment outcomes and toxicities.

Outcomes of Rural Men with Breast Cancer: A Multicenter Population Based Retrospective Cohort Study

Breast cancer is rare in men. This population-based study aimed to determine outcomes of male breast cancer in relation to residence and other variables. In this retrospective cohort study, men diagnosed with breast cancer in Saskatchewan during 2000-2019 were evaluated. Cox proportional multivariable regression analyses were performed to determine the correlation between survival and clinicopathological and contextual factors. One hundred-eight eligible patients with a median age of 69 years were identified. Of them, 16% had WHO performance status ≥ 2 and 61% were rural residents. The stage at diagnosis was as follows: stage 0, 7%; I, 31%; II, 42%; III, 11%; IV, 8%. Ninety-eight percent had hormone receptor-positive breast cancer. The median disease-free survival of urban patients was 97 (95% CI: 50-143) vs. 64 (46-82) months of rural patients (p = 0.29). The median OS of urban patients was 127 (94-159) vs. 93 (32-153) months for rural patients (p = 0.27). On multivariable analysis, performance status ≥ 2, hazard ratio (HR) 2.82 (1.14-6.94), lack of adjuvant systemic therapy, HR 2.47 (1.03-5.92), and node-positive disease, HR 2.32 (1.22-4.40) were significantly correlated with inferior disease-free survival in early-stage invasive breast cancer. Whereas stage IV disease, HR 7.8 (3.1-19.5), performance status ≥ 2, HR 3.25 (1.57-6.71), and age ≥ 65 years, HR 2.37 (1.13-5.0) were correlated with inferior overall survival in all stages. Although residence was not significantly correlated with outcomes, rural men had numerically inferior survival. Poor performance status, node-positive disease, and lack of adjuvant systemic therapy were correlated with inferior disease-free survival.

Abstract P4-02-12: Validation of Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) in early-stage invasive breast cancer patients of the Scottish Conservation Trial

Abstract Background: Adjuvant whole breast radiotherapy (RT) is provided to almost all women with early-stage invasive breast cancer after breast conserving surgery and appropriate systemic therapy. While there is increasing interest to personalize the use of RT based on molecular profiling, to date, there is no molecular signature available to reliably assess the benefit of radiotherapy after surgical resection. Here we assess the ability of a 16-gene signature named Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) to identify who may be suitable candidates for radiotherapy omission in patients of the Scottish Conservation Trial. Methods: The POLAR signature was applied to archival tissue from the Scottish Conservation Trial, which randomized 585 patients with stage I-II breast cancer, tumor size &amp;lt; 4 cm, and age ≤70 years old to receive RT or not. The archival tissue was measured for ER (ER+ &amp;gt;10%), PgR (PgR+ ≥20%), Ki67 (Ki67 high ≥14%), and HER2 (HER2+ defined as HER2 over-expressed or amplified). 26% received adjuvant chemotherapy, the remainder received tamoxifen 20 mg/daily for 5 years. Cox models for the locoregional recurrence (LRR) endpoint tested the association between treatment arms separately for patients with a low and high POLAR score using a pre-specified cut point. Cumulative incidences were computed, with distant metastasis and death without recurrence considered competing events. Results: 224 patients had tissue available and complete clinical data for analysis, 40 (18%) were node-positive. The distribution of clinicopathologic variables between the RT and no RT arms remained balanced. 43% were ER+/PgR+/Ki67 low/HER2-, 31% were ER+/HER2-/Ki67 high or PgR-, 5% were HER2+, and 13% were triple negative. The continuous standardized POLAR score was prognostic for LRR in the no RT arm after adjusting for relevant covariates (HR=1.78 [1.20-2.64], p=0.003). For patients with a POLAR-high score, the 10-year LRR rate was 31% [21%-42%] for patients not receiving RT and 8% [3%-15%] for patients receiving RT (HR 0.36 [0.19-0.69], p=0.0022). For patients with a POLAR low score, the 10-year LRR rates were 20% [10%-33%] for patients not receiving RT and 6% [1%-18%] for patients receiving RT; HR=0.28 [0.08-0.98], p=0.046). In the subgroup of node-negative patients with ER+/HER2-negative tumors (N=137), there was a statistically significant RT benefit for patients with a POLAR high score (HR=0.31 [0.11-0.88], p=0.028) but not for patients with a POLAR low score (HR=0.5 [0.1-2.4], p=0.39). Conclusions: For patients with early-stage invasive breast cancer treated with breast-conserving surgery without RT, POLAR is prognostic for LRR and may refine the selection of “low risk” for omission of RT. Citation Format: Karen J. Taylor, John MS Bartlett, John Bennett, S. Laura Chang, Bradley Arrick, Frederick Baehner, Joseph F. Loane, Tammy Piper, Elizabeth Mallon, Joanna Dunlop, Wilma J. Jack, Jacqueline Caldwell, Ian Kunkler, Linda J. Williams, Corey W. Speers, Felix Y. Feng, Lori Pierce, David A. Cameron. Validation of Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) in early-stage invasive breast cancer patients of the Scottish Conservation Trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-12.