Markers Of Response Research Articles

Abstract PR011: Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer

Abstract BCG immunotherapy is the standard of care for high-risk non-muscle invasive bladder cancer (NMIBC). The mechanisms of response to BCG are not fully understood. Patient response to BCG is highly variable with ~30-40% of individuals experiencing recurring or progressing disease after treatment within 24 months. Prognostic markers of response to treatment are not fully understood and remain insufficient for patient stratification. In order to develop an understanding of BCG response by comprehensive assessment of the tumor context. We performed single cell spatial proteomic profiling via imaging mass cytometry (IMC) on tissue microarrays of tumor biopsies from 53 bladder cancer patients (36 responding and 17 non-responding). We then validated our finding on an external cohort of whole pathology slides from 10 patients (4 responding and 6 non-responding). The IMC panel was developed based on scRNAseq profiles of bladder tumor samples to provide an unbiased cellular context of NMIBC. Additionally, IMC data is accompanied by hematoxylin and eosin (H&E) images of adjacent cuts and annotated by a clinical pathologist to distinguish tumor, immune, and stromal regions. We first developed a convolutional neural network (CNN) to reproduce pathologist annotations of tumor, stroma, and immune regions from H&E data with high accuracy (AUC>93%). We then registered IMC and annotated H&E images to train a random forest model to predict pathologist annotations using IMC data (AUC>95%). To determine the cellular composition of TMA spots, we used sparse non-negative matrix factorization (sNMF) to encode and cluster IMC data within tumor, stroma, and immune regions. NMF analysis revealed that a component defined primarily by KRT5, CD11b, and CD14, specifically within tumor-annotated regions adjoining COL1A1+ stromal regions, is a prognostic marker of poor response (p-value<0.05). This is consistent with previous findings that tumor-associated myeloid cells indicate poor prognosis. Additional NMF components of the immune annotated region showed weak associations with response. Immune regions defined by CD68, CD11b, CD31, and PDPN corresponded with poor BCG response, indicating myeloid-stromal-vascular interactions outside the tumor influence outcome, while vascular immune regions defined by high aSMA presence correspond with better response. Collectively, combining NMF components to generate a single response score for responding and non-responding patients (p-value<0.05) proved more effective at patient stratification than any single NMF component alone. The external cohort validated our random forest model for separating tumor, stroma, and immune regions, our sNMF model for clustering IMC images, and our response score (p-value<0.1). Citation Format: Ali Foroughi Pour, Dylan Baker, Santhosh Sivajothi, Bonnie Choy, Khyati Meghani, Yanni Yu, Leigh A. Fall, Benjamin Ristau, Joshua J. Meeks, Paul Robson, Jeffrey H. Chuang. Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR011.

Ultrasound-Guided Lidocaine Injection as a Novel Predictor of Response to Botulinum for Patients With Myofascial Pain Syndrome: A Case Report.

Botulinum injection is a well-known non-surgical intervention utilized in the management of myofascial pain syndrome (MPS). However, sparse evidence exists regarding the utility of ultrasound guidance of injectate or lidocaine as a predictive marker of patient response to botulinum toxin A (BTX-A). A 39-year-old male active duty service member demonstrated typical signs and symptoms of MPS. He reported a 10-year history of neck and back spasms that were triggered by exertion but also could occur spontaneously. Based on the characteristic regional motor-sensory defects, treatment options were discussed. With shared decision-making, the patient opted to try ultrasound-guided injection of lidocaine followed by xenomin brand BotoxA. Immediately following lidocaine injection, the patient reported complete relief of symptoms. Both injections were uncomplicated, and the patient reported great reduction in symptoms during the subsequent visit 2 months later. Relief of pain following ultrasound-guided injection of lidocaine may serve as an indicator of successful patient response to BTX-A in patients with MPS.

Integrated proteomic and genomic analysis to identify predictive biomarkers for valproate response in bipolar disorder: a 6-month follow-up study

BackgroundSeveral genetic studies have been undertaken to elucidate the intricate interplay between genetics and drug responses in bipolar disorder (BD). However, there has been notably limited research on biomarkers specifically linked to valproate, with only a few studies investigating integrated proteomic and genomic factors in response to valproate treatment. Therefore, this study aimed to identify biological markers for the therapeutic response to valproate treatment in BD. Patients with BD in remission were assessed only at baseline, whereas those experiencing acute mood episodes were evaluated at three points (baseline, 8 ± 2 weeks, and 6 ± 1 months). The response to valproate treatment was measured using the Alda scale, with individuals scoring an Alda A score ≥ 5 categorized into the acute-valproate responder (acute-VPAR) group. We analyzed 158 peptides (92 proteins) from peripheral blood samples using multiple reaction monitoring mass spectrometry, and proteomic result-guided candidate gene association analyses, with 1,627 single nucleotide variants (SNVs), were performed using the Korean chip.ResultsThe markers of 37 peptides (27 protein) showed temporal upregulation, indicating possible association with response to valproate treatment. A total of 58 SNVs in 22 genes and 37 SNVs in 16 genes showed nominally significant associations with the Alda A continuous score and the acute-VPAR group, respectively. No SNVs reached the genome-wide significance threshold; however, three SNVs (rs115788299, rs11563197, and rs117669164) in the secreted phosphoprotein 2 gene reached a gene-based false discovery rate-corrected significance threshold with response to valproate treatment. Significant markers were associated with the pathophysiological processes of bipolar disorders, including the immune response, acute phase reaction, and coagulation cascade. These results suggest that valproate effectively suppresses mechanisms associated with disease progression.ConclusionsThe markers identified in this study could be valuable indicators of the underlying mechanisms associated with response to valproate treatment.

Identification of collagen subtypes of gastric cancer for distinguishing patient prognosis and therapeutic response

AbstractBackgroundGastric cancer is a highly heterogeneous disease, presenting a major obstacle to personalized treatment. Effective markers of the immune checkpoint blockade response are needed for precise patient classification. We, therefore, divided patients with gastric cancer according to collagen gene expression to indicate their prognosis and treatment response.MethodsWe collected data for 1250 patients with gastric cancer from four cohorts. For the TCGA‐STAD cohort, we used consensus clustering to stratify patients based on expression levels of 44 collagen genes and compared the prognosis and clinical characteristics between collagen subtypes. We then identified distinct transcriptomic and genetic alteration signatures for the subtypes. We analyzed the associations of collagen subtypes with the responses to chemotherapy, immunotherapy, and targeted therapy. We also established a platform‐independent collagen‐subtype predictor. We verified the findings in three validation cohorts (GSE84433, GSE62254, and GSE15459) and compared the collagen subtyping method with other molecular subtyping methods.ResultsWe identified two subtypes of gastric adenocarcinoma: a high‐expression collagen subtype (CS‐H) and a low‐expression collagen subtype (CS‐L). Collagen subtype was an independent prognostic factor, with better overall survival in the CS‐L subgroup. The inflammatory response, angiogenesis, and phosphoinositide 3‐kinase (PI3K)/Akt pathways were transcriptionally active in the CS‐H subtype, while DNA repair activity was significantly greater in the CS‐L subtype. PIK3CA was frequently amplified in the CS‐H subtype, while PIK3C2A, PIK3C2G, and PIK3R1 were frequently deleted in the CS‐L subtype. CS‐H subtype tumors were more sensitive to fluorouracil, while CS‐L subtype tumors were more sensitive to immune checkpoint blockade. CS‐L subtype was predicted to be more sensitive to HER2‐targeted drugs, and CS‐H subtype was predicted to be more sensitive to vascular endothelial growth factor and PI3K pathway‐targeting drugs. Collagen subtyping also has the potential to be combined with existing molecular subtyping methods for better patient classification.ConclusionsWe classified gastric cancers into two subtypes based on collagen gene expression and validated these subtypes in three validation cohorts. The collagen subgroups differed in terms of prognosis, clinical characteristics, transcriptome, and genetic alterations. The subtypes were closely related to patient responses to chemotherapy, immunotherapy, and targeted therapy.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

Transdisciplinary Tactics and Prospects for Medical and Commercial Advancement in Kefir

Background: Kefir, a fermented milk beverage with a rich cultural heritage, has garnered significant attention due to its health benefits and commercial potential. This study explores the convergence of medical science, technology, and market dynamics to position kefir as a functional food with therapeutic applications. Objective: The study aimed to evaluate the health benefits of kefir, optimize its production processes, and investigate its therapeutic potential in chronic diseases and injuries. Methods: A randomized controlled trial was conducted with participants divided into kefir and control groups. Pre- and post-intervention health assessments were performed, measuring cardiovascular health, blood sugar levels, immune response markers, and neurological function. Biochemical analyses of blood samples were conducted to identify changes due to kefir consumption. Statistical analyses were performed using SPSS version 25. For process optimization, Response Surface Methodology (RSM) was applied to optimize fermentation conditions. Experimental studies included in vitro cell culture experiments and in vivo animal models to assess kefir’s effects on diabetes, Alzheimer's disease, and cancer. Microbial analysis was performed using genomic and proteomic techniques, and consumer sensory evaluations were conducted for new kefir formulations. Results: The kefir group showed a significant reduction in mean blood sugar levels from 96.62 mg/dL to 85.18 mg/dL (t-statistic=5.16, p=0.000004), while the control group showed no significant change. Optimized production conditions were determined to be a temperature of 25°C, fermentation time of 24 hours, and a grain-to-milk ratio of 0.1, achieving a quality score of 500. In the diabetes model, the kefir-treated group had a significant reduction in HbA1c levels (6.75) compared to the control group (7.41) (t-statistic=6.14, p=8.12×10^-8). For Alzheimer's disease, amyloid plaque deposition decreased significantly in the kefir group (37.34) versus the control group (50.13) (t-statistic=5.70, p=4.29×10^-7). In the cancer model, tumor growth was significantly reduced in the kefir group (64.18) compared to the control group (98.10) (t-statistic=7.45, p=5.09×10^-10). Microbial counts were highest and most stable under Condition A (11.05×10^7, SD=6.33×10^5). Consumer sensory evaluations of soymilk-based kefir resulted in a mean score of 3.1 (SD=1.32). Conclusion: Kefir demonstrates significant health benefits, including blood sugar regulation, and therapeutic potential in managing diabetes, Alzheimer's disease, and cancer. Optimized production conditions enhance its commercial viability. Future research should focus on long-term effects and real-world applications to validate these findings.

Galunisertib downregulates mutant type I collagen expression and promotes MSCs osteogenesis in pediatric osteogenesis imperfecta

Qualitative alterations in type I collagen due to pathogenic variants in the COL1A1 or COL1A2 genes, result in moderate and severe Osteogenesis Imperfecta (OI), a rare disease characterized by bone fragility. The TGF-β signaling pathway is overactive in OI patients and certain OI mouse models, and inhibition of TGF-β through anti-TGF-β monoclonal antibody therapy in phase I clinical trials in OI adults is rendering encouraging results. However, the impact of TGF-β inhibition on osteogenic differentiation of mesenchymal stem cells from OI patients (OI-MSCs) is unknown. The following study demonstrates that pediatric skeletal OI-MSCs have imbalanced osteogenesis favoring the osteogenic commitment. Galunisertib, a small molecule inhibitor (SMI) that targets the TGF-β receptor I (TβRI), favored the final osteogenic maturation of OI-MSCs. Mechanistically, galunisertib downregulated type I collagen expression in OI-MSCs, with greater impact on mutant type I collagen, and concomitantly, modulated the expression of unfolded protein response (UPR) and autophagy markers. In vivo, galunisertib improved trabecular bone parameters only in female oim/oim mice. These results further suggest that type I collagen is a tunable target within the bone ECM that deserves investigation and that the SMI, galunisertib, is a promising new candidate for the anti-TGF-β targeting for the treatment of OI.

Assessing the treatment response of lateral elbow tendinopathy using time-dependent ultrasonography, Doppler imaging, and elastography

ObjectiveTo investigate the structural alterations, neovascularity, and elasticity of tendons and the relationship between elasticity and the Patient Rated Tennis Elbow Evaluation score after undergoing US-guided fenestration or surgery in patients with chronic lateral elbow tendinopathy.MethodsParticipants from the per-protocol population of a randomized trial conducted between October 2016 and June 2020 were included. The surgery and fenestration groups included 24 (mean age, 50 ± 7 years [standard deviation], 10 men) and 29 (47 ± 8 years, 18 men) participants, respectively. Ultrasound exams were performed at baseline, 6 months, and 12 months. Statistical analyses included linear mixed effects and generalized equation estimation models.ResultsFenestration had no significant impact on tendon thickness (p = 0.46). Conversely, surgery significantly increased tendon thickness at 6 months (p < 0.0001) and remained elevated at 12 months (p = 0.04). Tendon echostructure exhibited a group effect (p = 0.03), indicating a higher proportion of pathological scores in the surgery group post-intervention compared to the fenestration group. Both groups showed a similar reduction in neovascularity from 6 to 12 months postintervention (p = 0.006). Shear-wave velocity increased in the fenestration group at 6 months (p = 0.04), while the surgery group experienced a nonsignificant decrease at 6 months, with some improvement at 12 months (p = 0.08). Changes in shear-wave velocity did not correlate with clinical outcome.ConclusionsFenestration and surgery reduced tendon neovascularity over time. Unlike surgery, fenestration did not impact tendon size while improving tendon echostructure and elasticity.Critical relevance statementFenestration and surgery equally alleviated symptoms and decreased tendon neovascularity in lateral elbow tendinopathy; however, fenestration did not alter tendon thickness and improved echostructure and shear-wave velocity, suggesting shear-wave velocity’s potential for quantitatively monitoring tendon elasticity during healing.Key PointsReliable markers for monitoring healing response and informing treatment protocols in elbow tendinopathy are lacking.Fenestration and surgery reduced tendon neovascularity, while fenestration improved tendon echostructure and shear-wave velocity.Shear-wave velocity may provide quantitative measures to monitor tendon elasticity in response to treatment.Graphical

Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury.

The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cells activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could impact nidogen-1 accessibility. Phosphoproteomics revealed that the 'AKI protector' Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin β1 to induce Wnts in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.

Analysis of Inflammatory Markers in Response to Induction of Reprometabolic Syndrome by a Eucaloric High Fat Diet in Normal Weight Women.

Obesity is associated with chronic low-level inflammation and is known to contribute to metabolic dysfunction and hypogonadotropic hypogonadism, which we have previously termed the 'Reprometabolic Syndrome.' To investigate potential factors involved in obesity-related reproductive endocrine dysfunction, we conducted a secondary analysis of inflammatory markers in a sample of normal weight women exposed to a one-month eucaloric high-fat diet (HFD), which, as reported earlier, induced the relative hypogonadotropic hypogonadism characteristic of Reprometabolic Syndrome. Eighteen healthy women with a BMI between 18.0-24.9kg/m2 and regular menstrual cycles participated in the study. Frequent blood sampling was performed during the early follicular phase before and after the one-month eucaloric HFD intervention (48% of calories from fat). Serum samples pooled from each participant were analyzed using immunoassay to measure levels of 30 cytokines, interleukins, and chemokines. Differences between pre- and post-HFD intervention measures were examined by one-sample t-tests. Exposure to the eucaloric HFD did not result in changes in body weight. HFD-induction of Reprometabolic Syndrome in normal weight women was associated with a significant elevation only in the anti- inflammatory cytokine IL-10 (p = 0.04). Eotaxin, IL-6 and MIP-1β also increased in response to the HFD, but not statistically significantly (p = 0.07). Results suggest that the increase in multiple inflammatory markers, typically associated with obesity, are not primary mediators of the relative hypogonadotropic hypogonadism of Reprometabolic Syndrome. Clinical Trials Registration Number: NCT02653092; Date of Registration: January 6, 2016.

Patterns of spontaneous neural activity associated with social communication abilities among infants and toddlers showing signs of autism.

Early disruptions to social communication development, including delays in joint attention and language, are among the earliest markers of autism spectrum disorder (autism, henceforth). Although social communication differences are a core feature of autism, there is marked heterogeneity in social communication-related development among infants and toddlers exhibiting autism symptoms. Neural markers of individual differences in joint attention and language abilities may provide important insight into heterogeneity in autism symptom expression during infancy and toddlerhood. This study examined patterns of spontaneous electroencephalography (EEG) activity associated with joint attention and language skills in 70 community-referred 12- to 23-month-olds with autism symptoms and elevated scores on an autism diagnostic instrument. Data-driven cluster-based permutation analyses revealed significant positive associations between relative alpha power (6-9Hz) and concurrent response to joint attention skills, receptive language, and expressive language abilities. Exploratory analyses also revealed significant negative associations between relative alpha power and measures of core autism features (i.e., social communication difficulties and restricted/repetitive behaviors). These findings shed light on the neural mechanisms underlying typical and atypical social communication development in emerging autism and provide a foundation for future work examining neural predictors of social communication growth and markers of intervention response.

Abstract PO5-13-08: Impact of systemic inflammation markers in response to neoadjuvant pembrolizumab and chemotherapy in triple-negative breast cancer - a retrospective analysis

Abstract Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment landscape for triple-negative breast cancer (TNBC). Systemic inflammation has been linked to cancer development and overall poor outcomes. Routine blood parameters have been investigated as potential inflammatory biomarkers in patients (pts) with various types of cancer, such as the derived NLR (neutrophils/(leucocytes-neutrophils) ratio (dNLR). A high dNLR has been associated with worse survival in several types of cancer. Specifically, it has been linked to lower pathologic complete response (pCR) rates after neoadjuvant chemotherapy (CTx). We investigated the association between systemic inflammation markers (dNLR, LIPI score and platelet-to-lymphocyte ratio [PLR]) and pCR in early TNBC pts treated with CTx + pembrolizumab (Pemb). Methods: Retrospective analysis of pts with early TNBC treated with Pemb + CTx from April 2022 to March 2023. dNLR was estimated from analytical values of peripheral blood collected before treatment (baseline). dNLR was calculated as the ratio of the absolute neutrophil number to the difference between absolute total leukocyte and absolute neutrophil counts. Univariate and multivariate logistic regression analyses were used to explore the association of dNLR with main clinical characteristics and dNLR capability (distributed as a continuous variable, using median cut-off) to predict pCR. Results: The study included 86 TNBC pts with a median age of 51 years (IQR: 42.2 – 56-7). Most pts had stage II disease (64%) and presented grade 3 tumours (n=66; 78.5%). HER2 low tumours represented 27% (n=23) of the cohort. Germline mutations were present in 20 pts (23%) with the following distribution: BRCA1 (n=14), BRCA 2 (n=5) and PALB2 (n=1). The median stromal tumor infiltrating lymphocytes (TILs) was 20% (IQR: 10-30%). The median Ki67 was 70% (IQR:50%-80%). All pts received Pemb. The majority of pts received carboplatin plus paclitaxel, followed by EC90 (n=77 (90%), while 10% (n=9) did not receive anthracyclines. Data concerning surgical outcomes was available in 80 pts. Pathological complete response rate was 61.2% (n=49). Pts achieving pCR presented significantly higher TILs (p&amp;lt; 0.01). Residual cancer burden (RCB) class (n=80) was distributed as follows: 0: 61% (n=49); I: 13% (n=10); II: 20% (n=16); III: 1% (n=1) and non-assigned: 5% (n=4). In pts with germline mutations (n=20), the pCR rate was 85% (n=17). Median dNLR at baseline was 1.30 (interquartile range (IQR): 0.99 – 1.86). High baseline dNLR levels considered a continuous variable was associated with a higher likelihood of achieving a pCR in univariate (OR: 2.59; 95% CI: 1.01–6.6; p-value = 0.046) but not in multivariate (OR: 2.33; 95%; CI: 0.85–6.33; p-value = 0.097) logistic regression analysis (adjusted for stage and histologic grade). High baseline dNLR, defined by the median cut-off, was associated with an increased likelihood of achieving pCR in both univariate (OR: 3.85; 95% CI: 1.47–10.1; p-value = 0.006) and multivariate (OR: 3.72; 95% CI: 1.28–10.8; p-value = 0.016) logistic regression analysis (adjusted for stage and histologic grade). Conclusion: In this real-life cohort of early TNBC pts treated with Pemb and CTx we found similar pCR rates (61.2%) to the ones reported in the KN522 trial. No association between PLR or LIPI score with pCR was evidenced. High baseline dNLR (assessed as a continuous variable or by the median cut-off) was associated with a higher likelihood of pCR in the univariate analysis. In the multivariate analysis, only high baseline dNLR defined by the median cut-off retained an association with pCR after stage and histologic grade adjustment. Despite several limitations, these exploratory data provide initial evidence of a potential association between dNLR values and independent prediction of pCR. Further research to assess the role of dNLR as a predictive marker for pCR in early TNBC pts treated with CTx + immunotherapy is needed. Citation Format: Martina Spotti, Elie Rassy, Alessandro Viansone, Fiona Pham, Layal Rached, Barbara Pistilli, Charles-Antoine Dutertre, Joana Mourato Ribeiro. Impact of systemic inflammation markers in response to neoadjuvant pembrolizumab and chemotherapy in triple-negative breast cancer - a retrospective analysis [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 PO5-13-08.

Eosinophilic granulocytes as a potential prognostic marker for cancer progression and therapeutic response in malignant melanoma.

The importance of eosinophilic granulocytes in cancer has been widely discussed in recent years. The current study reviews the evidence on the role of eosinophilic granulocytes in melanoma as a prognostic marker for cancer progression and the efficacy of treatment with modern immune checkpoint inhibitors. A total of 33 human clinical studies were included in the review, with heterogeneous data due to differences in patients populations, study design and inclusion of small study groups. However, 28 of the 33 studies suggested that eosinophilic granulocytes could be used as a prognostic biomarker for outcome and/or potential response to systemic treatment and/or occurrence of adverse events in melanoma patients. Nevertheless, the exact role of eosinophils remains to be elucidated. Further prospective, larger and better controlled studies are warranted to clarify the significance of eosinophilic granulocytes in patients with melanoma, in more details.

Abstract PO1-16-01: Clinicopathological and global transcriptional complexity of estrogen receptor low positive breast cancers in a contemporary Swedish prospective population-based cohort

Abstract Background: Estrogen receptor (ER) expression is currently the most consequential biomarker for prognostic and therapeutic decision making in clinical management of early breast cancer. Endocrine therapy (ET) is an important part of standard of care in most cases with ER-positive disease, and an effective therapy for patients with endocrine-responsive tumors, decreasing the risk of recurrence and improving the rate of survival. Unfortunately, ET comes with frequently bothersome side-effects and tend to decrease quality of life. The threshold to define ER positivity as a marker of endocrine responsiveness is non-uniform between international and local guidelines. Among ER low positive tumors (ERlow ,1-10% expression) endocrine responsiveness is uncertain. Swedish national guidelines recommend a cut-off of ≥10% for ER positivity and ET prescriptions, thus patients with ERlow tumors are often exempted from adjuvant ET. This study explores the clinicopathological characteristics, global transcriptional complexity and clinical outcome of ERlow tumors to better understand their biology and response to therapy. Methods: 9138 patients diagnosed with early breast cancer between 2010 – 2021 in Sweden with available clinicopathological data and RNA sequencing data were included. Patients were classified according to ER expression: ERneg (&amp;lt; 1%; n=897), ERlow (1-10%, n=158) and ERhigh ( &amp;gt;10%, n=8083). Adjuvant ET was provided to 3.6%, 16.8% and 91.5% of patients with ERneg, ERlow, and ERhigh tumors, respectively. Clinicopathological characteristics, overall survival (OS) and global transcriptional profiles of ERlow tumors were compared with ERneg and ERhigh tumors, respectively. Results: Generally, ERlow and ERneg tumor pathological characteristics were more similar to each other but were significantly distinct compared to ERhigh tumors. However, among patients with HER2-negative disease only, significant differences related to tumor biology persisted between ERlow compared with ERneg tumors; ERlow was enriched with tumors displaying a lobular histology, NHG grades 1&amp;2, PgRlow/high expression, low proliferation and Luminal &amp; HER2-enriched molecular subtypes (p&amp;lt; 0.05 for all comparisons). Moreover, multivariable survival analyses revealed that the risk of dying was significantly lower for patients with ERlow tumors compared to patients with ERneg (p=0.005) or ERhigh tumors (p=0.011) in this cohort. The distribution of clinicopathological characteristics and overall survival between ERlow compared with ERneg or ERhigh tumors were similar among patients with HER2-positive tumors only. Global transcriptional comparisons identified only 42 genes to be differentially expressed between ERlow vs ERneg tumors (FDR&amp;lt; 0.05); but gene set enrichment analyses failed to identify any significantly enriched cancer-related processes/pathways within the queried databases. Conclusions: Identification of optimal therapies for all subsets of breast cancer is necessary in the pursuit of personalized medicine. These results confirm that ERneg and ERlow tumors are pathologically and transcriptionally distinct from ERhigh tumors. Although subtle differences exists in the underlying biology of ERlow compared to ERneg tumors, similar therapeutic management excluding ET for patients with ERlow and ERneg disease is recommended in the Swedish context. Omission of adjuvant ET did not seem to compromise overall survival for ERlow relative to ERneg or ERhigh tumors, urging the need for prospective studies investigating the true benefit of ET for ERlow tumors. Our results also raise reasonable clinical thoughts on the benefits of new treatment strategies such as cyclin-dependent kinase 4/6 inhibitors, immunotherapy and antibody-drug conjugates for patients with ERlow expressing tumors. Citation Format: Siker Kimbung, Srinivas Veerla, Anna Ehinger, Johan Vallon-Christersson, Martin Malmberg, Niklas Loman. Clinicopathological and global transcriptional complexity of estrogen receptor low positive breast cancers in a contemporary Swedish prospective population-based cohort [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 PO1-16-01.

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.

Abstract PO2-28-03: E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis

Abstract Electronic cigarettes (E-cigs) have emerged as a significant public health concern due to their increasing popularity. These products have been associated with various harmful effects on human health. In the United States, breast cancer is the most diagnosed cancer among women, and its recurrence and metastasis are primary contributors to morbidity and mortality. Two critical factors in cancer progression are Epithelial-mesenchymal transition (EMT) and Cancer stem-like cells (CSCs), both of which play pivotal roles in metastasis. As a result, targeting the signaling pathways involved in these processes has become a promising avenue for cancer therapy. Autophagy, a cellular process, has dual roles in cancer development depending on the cancer type and stage. Given these considerations, our hypothesis centers on disrupting the EMT-mediated metastatic cascade in breast cancer by targeting autophagy in Cancer Stem Cells (CSCs). To investigate this hypothesis, we conducted experiments using mouse breast epithelial cells (HC11) and Triple-negative mouse breast cancer cells (4T1) exposed to E-cig aerosol at the air-liquid interphase (ALI) for 1 hr followed by a 24 hr recovery period. Our findings indicate that exposure to E-cig aerosols led to a decrease in epithelial markers (E-Cadherin and β-catenin) and an increase in mesenchymal markers (N-Cadherin and Vimentin) in HC11 cells. This suggests that normal cells may acquire characteristics associated with solitary migration. Furthermore, we observed significant changes in the levels of autophagy-related proteins (LC3B, Beclin-1, ATG12, and ATG16) and upregulation of stem cell markers (OCT4, SOX2, NANOG, KLF4, and c-MYC) in response to E-cig aerosol exposure, both in HC11 and 4T1 cells. Additionally, these alterations extended to changes in the expression of genes associated with the Transforming Growth Factor-beta (TGF-β) signaling pathway, known to play a potential role in promoting EMT. Our results provide a deeper understanding of the interplay between EMT, CSCs, and the autophagy process in the context of ALI exposure in normal and cancer cells. This underscores the need for further research into the shared molecular mechanisms of these three processes to identify common therapeutic targets that can simultaneously affect them. Citation Format: Shilpa Thota, Rizwana Begum, Naveen Chintalaramulu, Biplov Sapkota, Abhishek Pandit. E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis [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 PO2-28-03.

Abstract PO1-18-10: ANNE: A phase II single-arm clinical trial to assess the feasibility and efficacy of neoadjuvant anastrozole in patients with luminal breast cancer and low proliferative index in TNM stages II and III

Abstract Background: Neoadjuvant endocrine therapy (NET) is recommended for the treatment of invasive breast cancer (BC), particularly luminal subtypes, in locally advanced stages. Previous randomized studies have demonstrated the benefits of aromatase inhibitors (AIs) in this context. However, in clinical practice, NET is typically reserved for elderly or frail patients who may not tolerate neoadjuvant chemotherapy. Ideally, after identifying suitable candidates for NET, patients undergo tumor re-biopsy after 2 to 4 weeks of treatment, and only those with Ki67 ≤ 10% proceed with NET. Limitations of this strategy include the standardization of re-biopsy and the reliability of Ki67 immunohistochemical analysis, as treatment decisions are based on these results. Objective: To evaluate the feasibility and efficacy of NET in postmenopausal patients with stage II and III luminal BC and to identify predictive biomarkers of therapeutic response. The efficacy will be measured by the rate of patients with Ki67 ≤ 10% after 4 weeks and Preoperative Endocrine Prognostic Index (PEPI) score 0 on the surgical specimen. The feasibility of the study will be assessed by the acceptance rate of participating in the study (recruitment rate ≥ 50%) and the inclusion rate of &amp;gt;2 patients per month. Methods: This non-randomized phase II clinical trial includes postmenopausal women with invasive breast carcinoma, Scarf-Bloom-Richardson histological grades 1 or 2, Estrogen Receptor positive (Allred ≥ 6), progesterone receptor positive (any value), HER2 negative, Ki-67 antigen &amp;lt; 50% on immunohistochemistry, stages TNM II-III, with palpable tumor ≥ 2 cm, and ECOG 0-2. All eligible patients will receive anastrozole (1 mg/day) continuously until the day before the surgical procedure or exclusion from the study (in case of disease progression during endocrine therapy). After 2 to 4 weeks, a re-biopsy of the breast tumor will be performed. If Ki67 &amp;gt; 10%, endocrine therapy will be suspended, and the patient will be excluded from the study and redirected to neoadjuvant chemotherapy (routine care) or immediate surgery (as per attending clinical oncologist and surgeon). If Ki67 ≤ 10%, the patient will continue to receive NET. Extended NET will be administered for up to 10 months, with monthly physical examinations (using calipers) and bimonthly breast ultrasound. The presence of stable disease on ultrasound at 4, 6, and 8 months indicates scheduling surgery within 2 months thereafter, and disease progression at any time indicates surgery within one month. Patients will complete the EORTC QLQ-C30 and its supplemental module BR45 questionnaires every 2 months. Toxicity will be assessed using CTCAE version 4.0, and patients will respond to 11 specific items extracted from the Brazilian version of PRO-CTCAE. The radiological response will be evaluated by RECIST 1.1. Blood samples for molecular analyses will be collected at baseline and during re-biopsy. MicroRNA profiles will be evaluated using Nanostring, and extracellular vesicles will be examined using ATR-FTIR to identify predictive markers of therapeutic response (Ki67 &amp;lt; 10%) in re-biopsy. All cases will be evaluated using PAM50 at the initial biopsy and at surgery to determine the molecular phenotype (luminal A, B, or other) and molecular response (ROR score difference). Pathologic therapeutic response will be assessed using the PEPI score in all operated cases. The sample size was calculated to be 59 patients. The study was initiated in July 2022, and since then, a total of 26 patients have been enrolled. This study is registered with The Brazilian Registry of Clinical Trials (ReBEC): RBR-5pygzhj. Citation Format: Carlos Paiva, Izabella Oliveira, Vitor Guimarães, Alinne Faria, Domício Lacerda, Anapaula Uema, Nilton Onari, Bianca Paiva, Augusto Antoniazzi, Karina Oikawa, Maria Fernanda Machado, Matheus Godinho, Cristiano Souza, Chrissie Amirati, Gustavo Teixeira, Idam de Oliveira Jr, Márcia Marques, Yara Maia. ANNE: A phase II single-arm clinical trial to assess the feasibility and efficacy of neoadjuvant anastrozole in patients with luminal breast cancer and low proliferative index in TNM stages II and III [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 PO1-18-10.

Abstract PO1-24-11: Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast

Abstract Background: Ductal carcinoma in situ (DCIS) of the breast encompasses various subtypes with distinct behaviors. Immune cell infiltration within the tumor microenvironment is thought to influence DCIS progression. Tumor-infiltrating lymphocytes (TILs) serve as a surrogate marker for the adaptive immune response. The interaction between programmed death 1 (PD-1) and its ligand PD-L1 suppresses effector T-cell function. Major histocompatibility complex (MHC) class I molecules play a critical role in presenting tumor antigens to cytotoxic T cells. Despite the significance of PD-L1 and MHC class I in immune evasion, limited data exist on their correlation with T-cell infiltration in DCIS subtypes. Methods: Using immunohistochemistry, we examined MHC class I and PD-L1 expression, along with CD3+ and CD8+ T lymphocytes, in 131 DCIS cases through tissue microarrays. DCIS subtypes included hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2-), HR+/HER2+, HR-/HER2+, and triple-negative (TN) subtypes. Results: Among the 128 interpretable cases, the distribution was as follows: HR+/HER2- (50.8%), HR+/HER2+ (14.1%), HR-/HER2+ (26.5%), and TN (8.6%). HER2+ subtypes (HR+/HER2+ and HR-/HER2+) exhibited higher stromal TILs, CD3+ T cells, and CD8+ T cells compared to HR+/HER2- subtype. MHC class I loss was observed in 16.4% (21/128) of cases, with the highest frequency in the HR+/HER2- subtype (29.2%), followed by HR-/HER2+ (5.9%), HR+/HER2+ (0%), and TN (0%) subtypes. DCIS cases lacking MHC class I expression displayed lower stromal CD8+ T cell infiltration than those with intact MHC class I. PD-L1 expression in immune cells (≥1%) was detected in 18.8% (24/128) of cases and correlated with increased stromal TILs, CD3+ T cells, and CD8+ T cells. PD-L1 expression was more prevalent in HER2+ subtypes than in HR+/HER2- subtype. PD-L1-positive DCIS cases mostly retained MHC class I expression (95.8%), except for one case (4.2%) in the HR-/HER2+ subtype. The highest numbers of stromal CD3+ and CD8+ T cells were observed in DCIS cases with intact MHC class I and positive PD-L1 expression. The correlation between MHC class I and PD-L1 expression and infiltration of CD3+ and CD8+ T cells was maintained in HR+/HER2- and HR-/HER2+ subtypes. Notably, tumoral CD3+ and CD8+ T cells showed no significant association with MHC class I or PD-L1 expression. Furthermore, MHC class I and PD-L1 expression did not predict tumor recurrence. Conclusion: We observed distinct patterns of MHC class I and PD-L1 expression and T-cell infiltration among DCIS subtypes. Enhancing our understanding of MHC class I, PD-L1, and immune subsets, including CD8+ T cells, may contribute to the development of immune modulating therapies for DCIS, particularly in the HER2+ subtype. Citation Format: Nah Ihm Kim, Min Ho Park, Ji Shin Lee. Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast [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 PO1-24-11.

Abstract PO4-24-10: Prognostic role of SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in the locally advanced and metastatic breast cancer

Abstract Introduction: Breast cancer (BC) is associated with a continuous increase in incidence, with high mortality rates in several countries. There are four immunophenotypes of BC, luminal A, luminal B, triple negative (TN), and HER2+. Some proteins in tumor cells and the tumor microenvironment are part of tumorigenesis and metastases. The CD44 molecule is considered an important marker of the inflammatory response. CD44 is present in all leukocytes and on the surface of tumor stem cells, involving tumor invasion and metastasis. Some authors have demonstrated that the signal transducer and activator of transcription 3 (STAT3) and sex-determining region Y-related group box 2 (SOX2) are related to the regulation of somatic cell division and tumorigenesis and metastasis in BC. Aim: To evaluate SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in locally advanced and metastatic breast cancer. Methods: A cross-sectional study was conducted at the Hospital de Cancer de Pernambuco (HCP) and the Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) from March 2017 to April 2018. Sixty-five women diagnosed with breast cancer participated in this study, aged between 28 and 64. Of these, 51 women had stage III (locally advanced) breast tumors, and 14 with metastatic disease (stage IV). There were 24 healthy women aged 18 to 65 as a control group. The analysis of CD44, SOX2, and STAT3 expression was performed by flow cytometry. Results: elevated levels of CD44+/CD24neg cells and CD44+/CD24neg/SOX2+ cells were observed in the blood of BC patients with luminal B, HER2+, and TN compared to the control group (p&amp;lt; 0.0001). Low CD44+/CD24neg/STAT3+ levels in the patient's blood compared to the control group. Elevated CD44+/CD24neg and CD44+/CD24neg/STAT3+ cells were observed in locally advanced BC compared to metastatic group (p &amp;lt; 0.05). Patients with TNBC had elevated SOX2+T lymphocyte levels compared to luminal B and HER2+ subtypes groups (p&amp;lt; 0.05). HER2+ patients had low levels of T lymphocytes and SOX2+T cells compared to controls and luminal A groups (p &amp;lt; 0.05). High levels of total T cells (p=0.0007) and low SOX2+T lymphocytes (p=0.02) in locally advanced compared to metastatic disease (stage IV). Elevated levels of SOX2+T lymphocytes in patients with negative lymph nodes (N0) compared to N1 and N2 groups (p=0.004 and p &amp;lt; 0.0001, respectively). High levels of CD44+/CD24negSTAT3+ cells and T lymphocytes and low levels of STAT3+T lymphocytes in locally advanced TN subtype (stage III) compared to metastatic (stage IV). Conclusion: SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in peripheral blood are prognostic roles in breast cancer. SOX2 and STAT3 expression are potential predictive biomarkers of disease progression in breast cancer regardless of tumor subtype. Investigating these biomarkers in peripheral blood has some advantages because peripheral blood collection is less invasive, easy to access, and allows evaluation at diagnosis, during treatment, and in cases of recurrence. Citation Format: Denise Viana, Victor Andrade, Marcelo Salgado, Carolina Vasconcelos, Leuridan Torres. Prognostic role of SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in the locally advanced and metastatic breast cancer [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 PO4-24-10.

Abstract PO1-19-09: Integrating gene signatures to guide HR+ MBC therapy in a diverse cohort (INSIGHT)

Abstract Background: Black women with breast cancer (BC) have a 40% higher mortality rate compared to Non-Hispanic White (NHW) women. Among women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) BC, Black women have worse outcomes than NHW despite comparable systemic therapies. Gene expression profiling assays have been used in early-stage BC to provide prognostic and sometimes predictive information beyond standard immunohistochemical classifications. The 80-gene molecular subtype signature (BluePrint) with the 70-gene risk of distant recurrence signature (MammaPrint®) (Agendia, Irvine, CA) further classify HR+/HER2- BC into luminal A-type, luminal B-type, HER2-type, and basal-type tumors. Luminal B, HER2, and basal-type tumors (non-luminal A) are more aggressive with worse survival outcomes and are overrepresented among Black women. In the metastatic setting, the role of molecular subtype signatures in guiding therapeutic decisions has not yet been determined. We hypothesize that patients with metastatic HR+/HER2- non-luminal A-type BC post-progression on endocrine therapy +/- CKD4/6 inhibition will derive more benefit from chemotherapy than standard-of-care endocrine therapy in the second line setting. We also hypothesize that the impact of the intervention will be more pronounced in Black women compared to NHW women. Methods: This is a randomized phase II study that will evaluate the anti-tumor effect of capecitabine compared to physician’s choice endocrine therapy as second line therapy for adult ( &amp;gt;18 years) patients with non-Luminal A HR+/HER2- metastatic or unresectable locoregional invasive carcinoma (NCT 05693766). The study plans to enroll up to 62 patients. This trial enriches for racial/ethnic minority patients through collaborations with the University of Texas Southwestern and the University of Alabama at Birmingham, two health systems that serve a large minority population. Eligible patients who have received prior endocrine therapy with a CDK4/6 inhibitor will have their tumor tissue analyzed using the MammaPrint® and BluePrint assays. Patients with Luminal A tumors will receive standard of care and will be followed for survival only. Patients with non-Luminal A tumors will be randomized (1:1) to receive physician’s choice endocrine therapy versus capecitabine (dosed at 2000mg twice daily for 7 days on, 7 days off) with stratifications by molecular subtype and race. Patients will be evaluated for response every three cycles using the Response Evaluation Criteria in Solid Tumors. Therapy will be continued until evidence of disease progression or unacceptable major toxicity. The primary endpoint is progression free survival; secondary endpoints are overall response rate, clinical benefit rate, overall survival, and patient reported outcomes. The study will have 80% power to detect a minimal hazard ratio of 0.5 at one-sided significance level of 0.05. Cell-free DNA will be collected at baseline and at three times post-baseline to investigate potential genetic markers of disease response and resistance. Enrollment opened on June 1, 2023. We anticipate opening the trial at the external sites before the end of 2023. Citation Format: Moriah Forster, Jennifer Whisenant, Ben Park, Erica Stringer-Reasor, William Audeh, Andrea Menicucci, Fei Ye, Heather McArthur, Sonya Reid. Integrating gene signatures to guide HR+ MBC therapy in a diverse cohort (INSIGHT) [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 PO1-19-09.