I-SPY2 Trial Research Articles

Pexidartinib and standard neoadjuvant therapy in the adaptively randomized I-SPY2 trial for early breast cancer.

We investigated the small-molecule receptor tyrosine kinase-inhibitor of colony-stimulating factor-1 receptor pexidartinib in the stage II/III breast cancer in the I-SPY2 platform trial. I-SPY2 is an adaptive platform trial that features multiple arms of experimental agents administered on a background of standard neoadjuvant therapy with paclitaxel and adriamycin/cyclophosphamide, followed by definitive surgery. The adaptive randomization engine preferentially assigns patients based upon cumulative performance of each agent in a given breast cancer subtype based on hormone receptor and HER2 receptor status. The study endpoint is pathologic complete response. A total of 9 participants were randomized to receive pexidartinib with neoadjuvant paclitaxel before enrollment was halted due to a serious adverse event of vanishing bile duct syndrome. No participants received a full course of the study drug. Although there remains interest in agents targeting CSF-1, hepatic toxicity appears to be a limiting factor for their use in early breast cancer. NCT01042379 ( www. gov/ct2/show/NCT01042379 ).

Prediction of pathological complete response to chemotherapy for breast cancer using deep neural network with uncertainty quantification.

The I-SPY 2 trial is a national-wide, multi-institutional clinical trial designed to evaluate multiple new therapeutic drugs for high-risk breast cancer. Previous studies suggest that pathological complete response (pCR) is a viable indicator of long-term outcomes of neoadjuvant chemotherapy for high-risk breast cancer. While pCR can be assessed during surgery after the chemotherapy, early prediction of pCR before the completion of the chemotherapy may facilitate personalized treatment management to achieve an improved outcome. Notably, the acquisition of dynamic contrast-enhanced magnetic resonance (DCEMR) images at multiple time points during the I-SPY 2 trial opens up the possibility of achieving early pCR prediction. In this study, we investigated the feasibility of the early prediction of pCR to neoadjuvant chemotherapy using multi-time point DCEMR images and clinical data acquired in the I-SPY2 trial. The prediction uncertainty was also quantified to allow physicians to make patient-specific decisions on treatment plans based on the level of associated uncertainty. The dataset used in our study included 624 patients with DCEMR images acquired at 3 time points before the completion of the chemotherapy: pretreatment (T0), after 3 cycles of treatment (T1), and after 12 cycles of treatment (T2). A convolutional long short-term memory (LSTM) network-based deep learning model, which integrated multi-time point deep image representations with clinical data, including tumor subtypes, was developed to predict pCR. The performance of the model was evaluated via the method of nested 5-fold cross validation. Moreover, we also quantified prediction uncertainty for each patient through test-time augmentation. To investigate the relationship between predictive performance and uncertainty, the area under the receiver operating characteristic curve (AUROC) was assessed on subgroups of patients stratified by the uncertainty score. By integrating clinical data and DCEMR images obtained at three-time points before treatment completion, the AUROC reached 0.833 with a sensitivity of 0.723 and specificity of 0.800. This performance was significantly superior (p<0.01) to models using only images (AUROC=0.706) or only clinical data (AUROC=0.746). After stratifying the patients into eight subgroups based on the uncertainty score, we found that group #1, with the lowest uncertainty, had a superior AUROC of 0.873. The AUROC decreased to 0.637 for group #8, which had the highest uncertainty. The results indicate that our convolutional LSTM network-based deep learning model can be used to predict pCR earlier before the completion of chemotherapy. By combining clinical data and multi-time point deep image representations, our model outperforms models built solely on clinical or image data. Estimating prediction uncertainty may enable physicians to prioritize or disregard predictions based on their associated uncertainties. This approach could potentially enhance the personalization of breast cancer therapy.

A phenocopy signature of TP53 loss predicts response to chemotherapy

In preclinical studies, p53 loss of function impacts chemotherapy response, but this has not been consistently validated clinically. We trained a TP53-loss phenocopy gene expression signature from pan-cancer clinical samples in the TCGA. In vitro, the TP53-loss phenocopy signature predicted chemotherapy response across cancer types. In a clinical dataset of 3003 breast cancer samples treated with neoadjuvant chemotherapy, the TP53-loss phenocopy samples were 56% more likely to have a pathologic complete response (pCR), with a significant association between TP53-loss phenocopy and pCR in both ER positive and ER negative tumors. In an independent clinical validation in the I-SPY2 trial (N = 987), we confirmed the association with neoadjuvant chemotherapy pCR and found higher rates of chemoimmunotherapy response in TP53-loss phenocopy tumors compared to non-TP53-loss phenocopy tumors (64% vs. 28%). The TP53-loss phenocopy signature predicts chemotherapy response across cancer types in vitro, and in a proof-of-concept clinical validation is associated with neoadjuvant chemotherapy response across multiple clinical breast cancer cohorts.

The Immune-Related 27-Gene Signature DetermaIO Predicts Response to Neoadjuvant Atezolizumab plus Chemotherapy in Triple-Negative Breast Cancer.

We assessed the 27-gene RT-qPCR-based DetermaIO assay and the same score calculated from RNA sequencing (RNA-seq) data as predictors of sensitivity to immune checkpoint therapy in the neoTRIPaPDL1 randomized trial that compared neoadjuvant carboplatin/nab-paclitaxel chemotherapy (CT) plus atezolizumab with CT alone in stage II/III triple-negative breast cancer. We also assessed the predictive function of the immuno-oncology (IO) score in expression data of patients treated with pembrolizumab plus paclitaxel (N = 29) or CT alone (N = 56) in the I-SPY2 trial. RNA-seq data were obtained from pretreatment core biopsies from 242 (93.8%) of the 258 patients in the per-protocol-population. The DetermaIO RT-qPCR test, performed in the CAP/CLIA-accredited laboratory of Oncocyte Corp., was available for 220 patients (85.3%). A previously established threshold was used to assign DetermaIO-positive versus DetermaIO-negative status. Publicly available microarray data were used from I-SPY2. IO scores calculated from RNA-seq and RT-qPCR data were highly concordant. In neoTRIPaPDL1, DetermaIO-positive cancers (N = 92, 41.8%) had pathologic complete response (pCR) rates of 69.8% and 46.9% in the CT + atezolizumab and CT arms, respectively. In DetermaIO-negative cases, pCR rates were similar in both arms (44.6% vs. 49.2%; interaction test P = 0.04). PDL1 protein expression and stromal tumor-infiltrating lymphocyte count were not predictive of differential benefit from atezolizumab. In I-SPY2, IO-positive cancers (45.9%) had pCR rates of 85.7% and 16%, with and without immunotherapy, respectively. In IO-negative cancers, pCR rates were 46.7% versus 16.1%. DetermaIO identified patients who benefited from neoadjuvant immunotherapy resulting in improved pCR rate, independently of PDL1.

Implementation and impact of an electronic patient reported outcomes system in a phase II multi-site adaptive platform clinical trial for early-stage breast cancer.

We describe the development and implementation of a system for monitoring patient-reported adverse events and quality of life using electronic Patient Reported Outcome (ePRO) instruments in the I-SPY2 Trial, a phase II clinical trial for locally advanced breast cancer. We describe the administration of technological, workflow, and behavior change interventions and their associated impact on questionnaire completion. Using the OpenClinica electronic data capture system, we developed rules-based logic to build automated ePRO surveys, customized to the I-SPY2 treatment schedule. We piloted ePROs at the University of California, San Francisco (UCSF) to optimize workflow in the context of trial treatment scenarios and staggered rollout of the ePRO system to 26 sites to ensure effective implementation of the technology. Increasing ePRO completion requires workflow solutions and research staff engagement. Over two years, we increased baseline survey completion from 25% to 80%. The majority of patients completed between 30% and 75% of the questionnaires they received, with no statistically significant variation in survey completion by age, race or ethnicity. Patients who completed the screening timepoint questionnaire were significantly more likely to complete more of the surveys they received at later timepoints (mean completion of 74.1% vs 35.5%, P < .0001). Baseline PROMIS social functioning and grade 2 or more PRO-CTCAE interference of Abdominal Pain, Decreased Appetite, Dizziness and Shortness of Breath was associated with lower survey completion rates. By implementing ePROs, we have the potential to increase efficiency and accuracy of patient-reported clinical trial data collection, while improving quality of care, patient safety, and health outcomes. Our method is accessible across demographics and facilitates an ease of data collection and sharing across nationwide sites. We identify predictors of decreased completion that can optimize resource allocation by better targeting efforts such as in-person outreach, staff engagement, a robust technical workflow, and increased monitoring to improve overall completion rates. https://clinicaltrials.gov/study/NCT01042379.

Therapeutic response and outcomes with uncommon breast cancer subtypes in the I-SPY trial 2010-2022.

1 Background: Uncommon histologies are over-represented among high-risk breast cancer (BC) and denote an area with limited trial data and of significant unmet medical need. To better understand trial outcomes in this group and identify signals of tumor responsiveness, we report pathologic complete response (pCR) and early event-free survival (EFS) by disease subtype in the I-SPY2 trial. Additionally, the I-SPY2 trial currently utilizes a combination of tumor molecular signature and receptor status to determine response predictive subtype (RPS) first developed from 987 I-SPY2 patients [Wolf et al, Cancer Cell 2022]. We report disease response rates for those who received what is now known to be optimal RPS guided therapy. Methods: The I-SPY2 platform trial tests novel agents given neo-adjuvantly with a chemotherapy backbone in high-risk BC (HER2 positive, triple negative and high molecular risk estrogen receptor positive BC). Histologic images of research biopsies, local biopsy and surgical pathology reports were reviewed centrally by I-SPY pathologists. Receptor subtype distribution and pCR rates were summarized; and EFS was estimated using the Kaplan Meier method. Association between pCR and EFS was evaluated using the Cox proportional hazard model with significance assessed by the log rank test. Results: 144/2118 (7%) of I-SPY2 participants were identified with metaplastic (60), lobular (55), mucinous (9), micropapillary (8), neuroendocrine (4) and other (8) BCS. Tumor receptor status, pCR rate and EFS by tumor type are shown in the Table. For the full cohort, pCR was associated with better EFS (hazard ratio (95% CI): 0.12 (0.02 - 0.88), p = 0.01). Within metaplastic (metapBC) 11/32 (34%) and 5/28 (18%) patients had a pCR with or without checkpoint blockade, respectively. By RPS group, 9 of 18 (50%) (13 metapBC, 3 other, 2 lobular) in the HER2-Immune+ group who received RPS optimized therapy had a pCR. In this group 6/13 (46%) with metapBC had a pCR. In the HER2+ driven RPS groups, pCR rate in the HER+HER2orBasal was 88% (7/8) and 0% (0/3) in the HER2+Luminal. Conclusions: High pCR rates observed in metapBC and other among subsets of often difficult to treat BC subtypes support novel approaches and provide a roadmap for future study of uncommon BCs. Outcomes were improved when therapy matched RPS vulnerabilities. Participants presenting with uncommon BC subtypes will be prospectively identified in the I-SPY2.2 trial to further develop effective approaches for this group. Clinical trial information: NCT01042379 .[Table: see text]

Therapeutic response and outcomes with less common breast cancer subtypes in the I-SPY trial 2011-2022.

582 Background: Uncommon histologies are over-represented among high-risk breast cancer (BC) and denote an area with limited trial data and of significant unmet medical need. To better understand trial outcomes in this group and identify signals of tumor responsiveness, we report pathologic complete response (pCR) and early event-free survival (EFS) by disease subtype in the I-SPY2 trial. Additionally, the I-SPY2 trial currently utilizes a combination of tumor molecular signature and receptor status to determine response predictive subtype (RPS) first developed from 987 I-SPY2 patients (1). We report disease response rates for those who received what is now known to be optimal RPS guided therapy. Methods: The I-SPY2 platform trial tests novel agents given neo-adjuvantly with a chemotherapy backbone in high-risk BC (HER2 positive, triple negative and high molecular risk estrogen receptor positive BC). Histologic images of research biopsies, local biopsy and surgical pathology reports were reviewed centrally by I-SPY pathologists. Receptor subtype distribution and pCR rates were summarized. EFS was estimated using the Kaplan Meier method. Association between pCR and EFS was evaluated using the Cox proportional hazard model with significance assessed by the log rank test. Results: 144/2118 (7%) of I-SPY2 participants were identified with metaplastic (60), lobular (55), mucinous (9), micropapillary (8), neuroendocrine (4) and other (8) BCs. Tumor receptor status, pCR rate and EFS by tumor type are shown in the Table. For the full cohort, pCR was associated with better EFS (hazard ratio (95% CI): 0.12 (0.02 - 0.88), p = 0.01). Within metaplastic (metapBC) 11/32 (34%) and 5/28 (18%) patients had a pCR with or without checkpoint blockade, respectively. By RPS group, 9 of 18 (50%) (13 metapBC, 3 other, 2 lobular) in the HER2-Immune+ group who received RPS optimized therapy had a pCR. In this group 6/13 (46%) with metapBC had a pCR. In the HER2+ driven RPS groups, pCR rate in the HER+HER2orBasal was 88% (7/8) and 0% (0/3) in the HER2+Luminal. Conclusions: High pCR rates observed in metapBC and other among subsets of often difficult to treat BC subtypes support novel approaches and provide a roadmap for future study of uncommon BCs. Outcomes were improved when therapy matched RPS vulnerabilities. Participants presenting with uncommon BC subtypes will be prospectively identified in the I-SPY2.2 trial to further develop effective approaches for this group. 1. Wolf et al, Cancer Cell 2022. Clinical trial information: NCT01042379 . [Table: see text]

Impact of extent of disease at presentation on oncologic outcomes according to pathologic response to neoadjuvant systemic therapy among participants of the I-SPY2 clinical trial.

594 Background: Recent analyses in HER2+ and triple negative (TN) breast cancer (BC) treated with neoadjuvant systemic therapy (NAST) suggest that extent of disease at presentation (tumor [T] size and nodal [N] involvement) influences outcomes independently of response to NAST, even in the context of pathologic complete response (pCR). Given this, we evaluated the association of clinical T and N status prior to NAST with long-term outcomes in I-SPY2. Methods: We identified patients (pts) with TN and HER2+ BC who received NAST on the I-SPY2 trial with available clinical T size and N status prior to NAST, pathologic response after NAST, and follow up for recurrence and survival. We conducted univariable and multivariable (MV) Cox proportional models for Event Free (EFS), Distant Recurrence Free (DRFS), and Overall Survival (OS) in TN and HER2+ BC according to response after NAST, adjusting for residual cancer burden (RCB) category. Results: Of 1,170 pts with TN or HER2+ BC, 1,013 were eligible (TN: 623 and HER2+: 390), after 157 pts were excluded due to missing critical variables. Median follow up was 4.4 years (range 0.3-10.2). Median age was 48 for TN and 49 for HER2+ BC. Following NAST, pCR was achieved in 277 (45%) pts with TN and 197 (51%) pts with HER2+ BC. Among pts with pCR, separate MV analyses for TN and HER2+ BC showed no independent association between clinical T and N category with EFS, DRFS or OS (p values all &gt; 0.05). However, among pts with residual disease, clinical T category (but not clinical N status) prior to NAST retained independent prognostic information in both TN and HER2+ BC in a MV model adjusting for age, RCB class, and hormone receptor status (for HER2+ BC). Compared to cT1-2, cT3-4 stage at diagnosis was associated with worse EFS, DRFS, and OS in TNBC, and with worse EFS, DRFS (but not OS) in HER2+ BC (Table). Additionally, hormone receptor status was independently associated with outcomes in HER2+ BC with residual disease (data not shown). The 5y EFS for cT1-2 vs cT3-4 according to RCB class in TNBC were: RCB0: 94% vs 95%, RCB1: 90% vs 65%, RCB2: 74% vs 44% and RCB3: 45% vs 20%. The 5y EFS for cT1-2 vs cT3-4 according to RCB class in HER2+ were: RCB0: 95% vs 88%, RCB1: 89% vs 84%, RCB2: 85% vs 64% and RCB3: 56% vs 50%. Conclusions: Tumor size (but not N involvement) prior to NAST was independently associated with outcomes in TN and HER2+ BC with residual disease, independently of RCB class. TN and HER2+ BC achieving pCR exhibited favorable outcomes regardless of extent of disease prior to NAST. These data are consistent with prior findings of the Neobioscore contributing only in the context of residual disease. Clinical trial information: NCT01042379 . [Table: see text]

Pathological complete response to predict long term outcomes in the I-SPY2 TRIAL.

597 Background: The I-SPY2 platform trial uses pathologic complete response (pCR) to identify active agents in patients with stage 2/3 breast cancer. Only patients with HR+ HER2- tumors, and low risk 70-gene assay results are excluded. 2118 patients were enrolled from March 2010 through June 2022 with follow-up data from 2005 patients as of January 2024. We have reported the improved event-free (EFS) and distant-recurrence free (DRFS) survival for 950 patients (1) who achieved a pathological complete response (pCR). Here we report the outcomes for the additional 1055 patients not included in our initial report. Methods: Patients have been randomized to one of 25 arms. Residual Cancer Burden (RCB) assessment was performed by site pathologists who were trained to ensure uniform response classification; RCB 0 = pCR. EFS is defined as time to local or distant recurrence or death. DRFS is defined as time to distant recurrence or death. Patients without events were censored at time to last follow-up. Association between pCR and EFS/DRFS was assessed using Cox proportional hazard modeling with significance assessment using the log rank test. EFS and DRFS at 3 years are estimated using the Kaplan Meier method. Results: Median follow up was 3.3 years (0.05-10.2 y). There were 144 EFS and 123 DRFS events. pCR rates varied by subtype, but achieving pCR was associated with an EFS of 96% and DRFS of 96%. For patients without pCR the EFS was 82% and DRFS was 84% at 3 years. The relationship between pCR and EFS was significant and clinically impactful within each subtype. Conclusions: pCR continues to have a strong positive association with EFS and DRFS as in our initial report. It is notable that EFS for non-pCR patients improved to 82% from our initial report of 78%. Initially few patients received additional adjuvant therapy except for standard of care endocrine and HER2-targeted therapy. Increased adoption of post neoadjuvant therapy with the approval of several agents for patients with non-pCR likely contributed to this improvement. I-SPY also reported a shift in residual cancer burden for patients receiving investigational therapies, which also may improve outcome for non-pCR patients. Taken together, these data demonstrate the value of pCR as an early endpoint regardless of tumor type or drugs delivered. These findings emphasize the need for new agents that will allow all patients to achieve pCR and potentially avoid the need for additional therapy after surgery. 1. Yee, et al. JAMA Oncol 2020. 2. Symmans, et al. JAMA Oncol 2021. Clinical trial information: NCT01042379 . [Table: see text]

Correlation of hormone receptor positive HER2-negative/MammaPrint high-2 breast cancer with triple negative breast cancer: Results from gene expression data from the ISPY2 trial.

573 Background: A subset of Hormone Receptor positive (HR+), HER2 negative (HER2-) breast cancers (BCs) classified by the MammaPrint (MP) assay as High-2 (MP-H2) show clinical behavior similar to triple-negative (TN) BC. The aim of this analysis was to assess molecular similarities between the HR+/HER2-/MP-H2 (HR+/MP-H2), HR+/HER2-/MP-High-1 (HR+/MP-H1), and TN/MP-H2 (TN/MP-H2) BCs. Methods: Gene expression data used for this analysis [GEO GSE194040] was derived from pre-treatment samples of the I-SPY2 trial, which is an ongoing multicentric randomized Phase II neoadjuvant platform trial for high-risk stage II or III BC. Six hundred eighty-four pretreatment samples were included in this analysis: 274 corresponded to HR+/MP-H1, 105 to HR+/MP-H2, and 305 to TN/MP-H2, accounting for 7 experimental plus a control arm. We used principal component and distance analyses, differentially expressed genes (analysis performed for pairwise comparisons of the 3 subtypes, a linear model, log fold-change and moderated-t p-values relative to a minimum fold-change threshold of fc = 1.5), gene set enrichment analysis using KEGG and GO, and Ingenuity Pathway Analysis, as well as 32 previously defined qualifying biomarkers to study similarities in biology between the subgroups. Results: On principal component and distance analyses, results reveal close similarity between HR+/MP-H2 and TN/MP-H2 indicating shared transcriptomic features. Gene expression analysis revealed that HR+/MP-H2 tumors resemble TN/MP-H2 more than HR+/MP-H1 cancers. Differential gene expression analysis revealed that HR+/MP-H2 and TN/MP-H2 cancers presented only two differentially expressed genes, compared to HR+/MP-H1 and HR+/MP-H2, which presented 577 differentially expressed genes. Upon pathway analysis, HR+/MP-H1 cancers presented the most significant upregulation in the glucocorticoid receptor signaling, DHCR24, and HOTAIR pathways, compared to HR+/MP-H2 cancers. Furthermore, HR+/MP-H2 and TN/MP-H2 shared enrichment of DNA repair and cell cycle pathways. In addition, various biomarkers were analyzed, and among the most representative shared biomarkers for HR+/MP-H2 and TN/MP-H2 were immune features, higher proliferation, and a lower expression of ESR1 co-expressed genes as compared to HR+/MP-H1 cancers. The rates of pathologic complete response were 41% for TN/MP-H2, 31% for HR+/MP-H2, and 11% HR+/MP-H1 cancers among all included patients. Conclusions: A subset of HR+ cancers defined by MP-H2 status closely resemble TN in molecular and clinical features. The similarity of pCR rates for MP-2 observed within either HR+/HER2- and TN is supported by our findings of similarity of active biology between HR+/MP-2 and TN/MP-2. These findings have implications for understanding the biology of HR+/MP-H2 BCs and optimizing treatment strategies.

Incidence and time to onset of immunotherapy-related adrenal insufficiency in the I-SPY2 trial.

584 Background: I-SPY2 was the first randomized trial to demonstrate an improvement in pCR and EFS when Immune Checkpoint Inhibitors (ICI) were added to standard neoadjuvant chemotherapy. This finding was validated in the phase 3 KEYNOTE 522 trial for triple-negative breast cancer, leading to approval of pembrolizumab (pembro) in this setting. ICIs add the risk of immune-related adverse events (irAEs); while most irAEs are reversible, endocrinopathies appear to persist. Higher rates of adrenal insufficiency (AI), either from primary adrenalitis or hypophysitis, have been reported with ICI-based therapy (tx) in early breast cancer (EBC) compared to advanced disease. Here we describe the incidence and time of onset of AI with ICI-based tx in I-SPY2. Methods: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes defined by hormone receptor, HER2, and MammaPrint status to evaluate novel agents in the neoadjuvant setting in patients (pts) with high-risk EBC. Treatment included weekly paclitaxel (T) plus ICI-based tx x 4 cycles, followed by doxorubicin/cyclophosphamide (AC) every 2-3 weeks x 4. 6 completed investigational arms included ICIs: T+pembro x 4, T+pembro x 8 (+/- AC), T+pembro+SD101 (TLR9 agonist), T+durvalumab+olaparib, T+cemiplimab (cemi), and T+cemi+REGN3767 (anti-LAG-3). AI diagnoses were made by treating providers with review from two board certified endocrinologists. AI was defined as AM serum cortisol level &lt; 3 mcg/dl in absence of recent glucocorticoid treatment. If ACTH was low or normal, pt was dx with hypophysitis; if ACTH was elevated, pt was dx with primary AI. Results: 442 pts in I-SPY2 received ICI-based tx; 11.8% (n=52) developed AI (primary+hypophysitis). Mean age of pts who developed AI was 47.3 vs 48.6 yrs in those who did not (p=0.44). Incidence of AI was highest in arms with dual immune modulation (18.1%) as compared to single ICI tx (8.5%). Median time to onset varied by arm, ranging from 92-204 days from first ICI dose. While some cases occurred during ICI tx (13.5%), the majority occurred between 12-24 wks after tx initiation, with 21.2% diagnosed after completion of all tx. The majority (94.2%) of pts with AI were symptomatic; the most common presenting symptoms included fatigue (59.6%), nausea (48.1%), and vomiting (34.6%). All pts with AI remain on glucocorticoid replacement tx. Conclusions: The incidence of AI in EBC is higher than has previously been reported in advanced disease, with higher rates observed in those tx with dual immune modulation. No correlation was observed with age; correlation of AI with response predictive subtype and other clinicopathologic features is ongoing and will be presented. Given the high incidence of AI in EBC, close monitoring in the peri/postoperative setting and education of pts and providers is critical, as risk persists even after completion of tx. Work to identify risk factors for AI is ongoing. Clinical trial information: NCT01042379 .

Association of germline polygenic risk for thyroid autoimmunity with overall survival in the I-SPY2 Trial.

10600 Background: Germline genetic variation influences immune activity and risk of autoimmunity, and has been hypothesized to play a role in response to cancer therapeutics and survival. Prior studies of breast cancer have reported associations between genetic variation in thyroid autoimmunity and overall survival (OS). Here, we aim to investigate the association between a polygenic risk score for hypothyroidism (PRS-H) and response to therapy in women with early-stage breast cancer, and hypothesize that high PRS-H will be associated with better treatment response and survival. Methods: This study includes 1,368 women aged 18 years and above with stage II-III breast cancer of size &gt; 2.5cm by clinical exam, with germline data available. It is an approved biomarker analysis of the I-SPY 2 trial, which is a phase II, adaptive, randomized platform trial across multiple sites studying novel cancer therapeutics across multiple arms for use as neoadjuvant chemotherapy for early-stage breast cancer. The outcome of survival is defined as alive at the most recent follow-up. Using the Cox proportional hazards model, we evaluate the association between a previously published PRS-H and overall survival. Models are adjusted for age at screening, immunotherapy status, and five principal components. Results: Among 1,368 early-stage breast cancer women, 33% (n=447) had pCR, and 85% (n=1,157) were alive at the last follow-up. Additionally, 16% (n=219) of women received immunotherapy. We observe a significant association between PRS-H and OS (HR = 0.78, 95% CI 0.66-0.92, p=0.004). Individuals in the top 10th percentile of the PRS-H (highest genetic risk) have improved survival (log rank p=0.04). Conclusions: Germline genetic variation in thyroid autoimmunity proclivity is associated with improved overall survival in our cohort of women with early-stage breast cancer receiving neoadjuvant chemotherapy. Our findings suggest that the genetic risk for autoimmunity may be important for long-term survival. We will expand this analysis to include the entire I SPY 2 population when all patients have completed genotyping. We plan to further characterize the role of germline autoimmunity profiles and survival. Clinical trial information: NCT01042379 .

Characterizing the 70-gene MammaPrint risk stratification in Chinese luminal type breast cancer and its correlation with clinicopathological characteristics, immune and metabolic functions.

e12559 Background: The 70-gene MammaPrint is endorsed by the NCCN guidelines for predicting 5-year distant metastasis risk in HR+, HER2-, with 0-3 lymph nodes, early invasive breast cancer. Similarly, it is recommended by the CSCO breast cancer guidelines for prognostic assessment post breast cancer surgery. However, no prior studies have investigated its application in Chinese luminal-type breast patients or its correlation with immune and metabolic functions. Methods: This analysis encompasses 76 early-stage invasive Luminal breast cancer patients, registered at the First People's Hospital of Foshan between 2020 and 2023, with recorded clinical characteristics under informed consent. Patient-reported data include clinical features, pathology, and the 70-gene MammaPrint risk of recurrence. Additionally, RNA-seq analysis was conducted in 24 patients. Results: Among 76 enrolled patients,55.3% were categorized as MammaPrint low risk (MP-Low), and 44.7% as MammaPrint high risk (MP-High). Patients over 50 were more likely MP-Low (p=0.028, OR=2.860), while those with &gt;20% Ki67-stained cells were prone to MP-High (p&lt;0.001, OR=0.160). Comparing with MINDACT, there was 52.3% consistency, with 55.6% of MP-Low patients being C-Low (MINDACT risk Low) and 47.1% of MP-High patients being C-High (MINDACT risk High). Notably,21% of C-Low patients showed MP-High risk, and 27% of C-High patients exhibited MP-Low risk, of which 83.3% were ≥50 years old. Based on MINDACT and ISPY2 trial outcomes, patients were further categorized into four groups: MP-Ultralow, MP-Low but not ultralow (LNUL), MP-High but not ultrahigh (HNUH) and MP-Ultrahigh. The study identified an 18% MP-Ultralow population, suggesting that excessive adjuvant therapy might be avoided in these patients. Additionally, 41% of patients were identified as LNUL group, indicating potential benefit from adjuvant treatment or endocrine prolongation therapy. 4% were identified as MP-Ultrahigh group, suggesting potential benefits from neoadjuvant targeted therapy, albeit a poorer long-term benefit in the non-pCR portion. Further transcriptome analysis in 24 patients revealed significantly higher levels of Th2 cells in the MP-High group, with no significant difference observed in CD8 T cells between the two groups. Meanwhile, metabolic-related signatures also differed, with significantly higher levels of Dopamine, Norepinephrine, Heme, and Epinephrine biosynthesis in the MP-Low group. Conclusions: This study elucidates the intricate relationships between MammaPrint prediction results of 70 genes and clinical characteristics, immune and metabolic signatures in Chinese Luminal type breast cancer patients. The refined risk classification provides valuable insights for precision treatment, advancing personalized therapeutic strategies.

Harnessing cell-free orphan non-coding RNAs as a predictive measure of long-term survival in neoadjuvant breast cancer therapy.

585 Background: Pathologic complete response (pCR) is a valuable metric for predicting survival following neoadjuvant therapy (NAT) in breast cancer; however, blood-based biomarkers may refine the prediction of treatment outcomes and inform follow-up treatment decisions. Orphan non-coding RNAs (oncRNAs) are a novel category of small RNAs that are present in tumors and largely absent in healthy tissue. Here, we explore the feasibility of a tumor-naive oncRNA-based liquid biopsy assay for predicting distant recurrence-free survival (DRFS) in breast cancer patients using a standard blood draw. Methods: Our cohort included 538 high-risk breast cancer patients from the multicenter I-SPY2 trial having serum available after NAT and before surgery (timepoint T3). Of the 538 patients (mean age: 48.8 ± 10.3), 191 (36%) achieved pCR. We isolated small RNA from 0.8 mL of patient serum and sequenced it at an average depth of 50 million 100-bp single-end reads. We used our catalog of tumor-derived oncRNAs, discovered in The Cancer Genome Atlas, to develop an AI model for predicting tumor burden based on tumor size. Our AI model was trained on oncRNA profiles from an independent cohort of 719 serum samples collected at diagnosis from individuals with cancer. We assessed tumor burden by oncRNA in the I-SPY2 cohort at T3 and associated this measure with DRFS using robust cutpoints established for each of the two pCR groups: high- ( n=70) and low-burden ( n=468) cutpoints were verified through cross-validation and almost all folds converged on a single value, which we used in our analysis. Results: The continuous oncRNA-derived tumor burden at T3 had a hazards ratio (HR) of 2.4 (95% CI: 1.1–5.1) for predicting DRFS. This measure remained significant in multivariate Cox models that included pCR (HR = 2.7 [1.3–5.6]) or residual cancer burden (RCB II/III versus 0/I; HR = 3.4 [1.6–7.1]). Patients with pCR had a 3-year DRFS of 94%; patients with pCR having low ( n=180) versus high ( n=11) oncRNA burden resulted in 95% and 73% 3-year DRFS, respectively (HR = 7.4 [2.3–24]). Those without pCR had a 3-year DRFS of 78% and stratification by oncRNA burden resulted in 85% and 71% 3-year DRFS for the low ( n=288) and high ( n=59) burden groups, respectively (HR=2.4 [1.5–3.9]). Importantly, we saw no restricted mean survival time difference (RMST) at 3 years between those with and without pCR among the high oncRNA burden group (0.1-month difference, p=0.97). Conclusions: We demonstrate the utility of a tumor-naive, oncRNA-derived tumor burden AI model as a predictive measure of treatment outcome and as a prognostic biomarker that can be used in addition to pCR to further stratify patients as low or high risk of recurrence. Integration of our oncRNA assay with existing biomarkers and clinicopathologic variables, has the potential to add value for predicting outcomes and prioritizing patients that would benefit from additional care.

Abstract PO1-15-09: Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- Breast Cancer

Abstract Background: Immune checkpoint blockade (ICB) has substantially improved patient overall and progression-free survival in triple-negative breast cancer (TNBC), but its efficacy remains to be elucidated in the hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer, which is characterized by generally impotent lymphocytic infiltration. In the I-SPY2 trial (NCT01042379), pembrolizumab- or durvalumab-containing neoadjuvant therapy increased the pathologic complete response of HR+/HER2- patients. However, pembrolizumab-containing therapy failed to improve the survival of metastatic HR+/HER2- breast cancer patients (NCT03051659). These inconsistent results underscored the necessity of the investigation of predictive biomarkers to pinpoint potential ICB responders in HR+/HER2- breast cancer. Method: In this study, we collected pretreatment tumor samples from 16 patients in two respective clinical trials (NCT: 04215003 and 04355858) and profiled droplet-based single-cell sequencing, bulk RNA-Seq. Patients were treated with camrelizumab (PD-1 inhibitor)-containing therapy, and five of them achieved partial relief after an 8-week course. We also leveraged the bulk RNA-Seq data from the I-SPY2 trial and Fudan University Shanghai Cancer Center (FUSCC) TNBC ICB trials (NCT04418154 and NCT04613674) for external validation. Another FUSCC transcriptome dataset consisting of 933 treatment-naïve HR+/HER2- or TNBC breast cancer patients was used to detect the abundance of cell types. In vitro and in vivo experiments were used to explore mechanisms of anti-tumor immune response. Results: Based on the single-cell sequencing results, we comprehensively delineated the microenvironmental landscape of HR+/HER2- breast cancer. We discovered responders of immunotherapy showed a higher presence of CXCL9/10+ M1-like macrophages and exhibited enrichment of tumor cells with ferroptosis. We further identified the upregulation of vitamin A metabolism and the vitamin A metabolic gene CRABP1 in tumor cells with ferroptosis. We verified the vitamin A metabolism could induce ferroptosis in HR+/HER2- breast cancers rather than TNBC by CRABP1-ERK axis, which could further recruit CXCL9/10+ M1-like macrophages and promote anti-tumor immune response in HR+/HER2- breast cancer. Combining anti-PD1 with enhancing vitamin A metabolism via retinoid acid possessed greater therapeutic efficacy than monotherapy in HR+/HER2- breast cancers. Finally, we developed an immune therapy response score and validated its reliability in predicting the immunotherapy efficacy for HR+/HER2-breast cancers through both internal and external cohorts. Conclusion: Our study represents the earliest efforts to decipher the microenvironmental landscape and the mechanisms underlying the response to ICB in HR+/HER2- breast cancer at single-cell resolution. We unraveled a HR+/HER2- breast cancer-specific Vitamin A-ferroptosis axis that mediated immunotherapy response and constructed a robust immunotherapy efficacy prediction score based on this Vitamin A-ferroptosis axis. Citation Format: Yi-Fan Zhou, Hu-Yun-Long Zhang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- 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 PO1-15-09.

Abstract PO5-15-04: MammaPrint index predicts neoadjuvant chemosensitivity in patients with HR+HER2- early-stage breast cancer in the real-world evidence FLEX study

Abstract Background: Neoadjuvant chemotherapy (NCT) yields low pathologic complete response (pCR, ypT0/is ypN0) rates in hormone receptor positive (HR+), HER2- early-stage breast cancer (ESBC). Genomic signatures may predict chemosensitivity and treatment benefit better than receptor status based clinical subtyping. The 70-gene expression signature, MammaPrint (MP), classifies patients with ESBC as having a High or Low Risk of distant recurrence. In the NBRST and ISPY2 trials, further stratification of MammaPrint High Risk into High 1 (H1) or High 2 (H2) showed significantly higher pCR rates to NCT or targeted agents in MP H2 cancers compared to MP H1 tumors. We evaluated MammaPrint H1 and H2 status as a biomarker for neoadjuvant chemosensitivity in patients with HR+HER2- ESBC enrolled in the real-world evidence FLEX study. Methods: FLEX (NCT03053193) is an ongoing prospective, observational trial that has currently enrolled 12,328 patients with ESBC who were tested with MammaPrint as standard of care, with or without molecular subtyping signature, BluePrint (BP), and consented to clinically annotated full genome data collection (data locked Feb. 2023). Patients with HR+HER2-, MP High Risk tumors who received NCT (majority received AC-T) and had pCR data available were included in this analysis (n = 214). Patients were stratified into MP H1 (index 0.000 to -0.569) and H2 (index -0.570 to -1.000) groups. BP classified MP High Risk tumors into Luminal B-, HER2-, or Basal-Type. Differences in clinical characteristics and pCR rates between H1 and H2 tumors was assessed by Chi-Squared test and two-sided proportional z-test, respectively. The association between MP H1 and H2, BP subtype, and pCR was assessed using logistic regression and was adjusted for age, race, grade, T stage, N stage, and NCT regimen. Results: MammaPrint classified 142 (66%) cancers as H1 and 72 (34%) as H2 tumors. Age, menopausal status, race, tumor stage, and lymph node status were comparable between both groups. Although most H2 tumors (78%) were Grade 3, only 59% of all Grade 3 tumors were H2. Nearly all (98%) H1 tumors were Luminal B whereas for H2 tumors, 51% were Luminal B and 49% were Basal by BP classification. pCR rate was significantly higher in H2 tumors (29.2%) compared to H1 tumors (6.3%, p &amp;lt; 0.01) (Table). Basal-Type, H2 tumors (n=35) exhibited the highest pCR rate of 37.1%. Among BP Luminal B tumors, those with MP H2 tumors had a significantly higher pCR rate (21.6%) vs. MP H1 tumors (5.8%, p = 0.003). Multivariate analysis revealed MP H2 (OR=4.91, p=0.003) and BP Basal-Type (OR=3.54, p=0.03) were significantly associated with likelihood of pCR, whereas clinical variables were not associated with pCR. Conclusion: These data demonstrate MammaPrint and BluePrint utility to predict the likelihood of achieving pCR after NCT in HR+HER2- ESBC. Although both MP High Risk groups exhibit chemosensitivity, High 2 tumors have higher chemosensitivity than High 1 tumors. MP High 2 status can be utilized to identify ER+ patients who are the most likely to experience pathologic downstaging and pCR after NCT. Also, patients with High 2 tumors treated with PD-L1 or PARP inhibitors in the ISPY2 trial exhibited significantly higher pCR rates than those observed for High 2 tumors treated with NCT alone. These data suggest that for patients with MP High 2 tumors, neoadjuvant use of chemotherapy is appropriate and these patients may further benefit from the addition of targeted agents to standard NCT. Table: Distribution of pCR rates among MammaPrint H1 and H2 and BluePrint molecular subgroups Data presented as # of patients with pCR/(total # patients in subgroup) and as percentage Citation Format: Joyce O'Shaughnessy, Lajos Pusztai, Cathy Graham, Pat Whitworth, Peter Beitsch, Cynthia Osborne, Rakhshanda Rahman, Andrea Menicucci, William Audeh, FLEX Investigators' Group. MammaPrint index predicts neoadjuvant chemosensitivity in patients with HR+HER2- early-stage breast cancer in the real-world evidence FLEX study [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 PO5-15-04.

Abstract PO4-02-03: Distribution of MammaPrint, BluePrint, and Response Predictive Subtypes based on ImPrint and Reprint in ER+/HER2- Invasive Lobular Carcinoma – A FLEX sub study

Abstract Background: Invasive lobular carcinoma (ILC), the second most common histologic subtype of breast cancer, is increasingly recognized as a distinct tumor type compared to the more common invasive ductal carcinoma (IDC). While ILC is known to have lower rates of pathologic complete response to neoadjuvant chemotherapy, ILC tumors are biologically heterogenous. As such, genomic signatures might identify patients with ILC who might benefit from tailored treatment options. The gene expression signature MammaPrint (MP) classifies tumors as having a Low Risk or High Risk of distant recurrence. MP combined with BluePrint (BP), a molecular subtyping signature, categorizes tumors as Luminal A (MP Low Risk), Luminal B (MP High Risk), Basal or HER-2 type. ImPrint is a 53-gene signature that identifies HR+HER2- patients predicted to benefit from immune checkpoint inhibitors. RePrint is a 60-gene DNA repair deficiency (DRD) signature that identifies HR+HER2- patients who may benefit from PARP inhibitors with platinum agents. Response Predictive Subtypes (RPS), used in the I-SPY2 trial, combine clinical subtype and these genomic signatures to personalize treatment planning and improve outcomes. In this study, we determined the distribution of 3 RPS in HR+HER2-: (1) ImPrint-/RePrint-, (2) ImPrint-/RePrint+ (3) ImPrint+, in patients ILC compared to IDC enrolled in the FLEX trial. Methods: This study includes 1078 HR+HER2-women with ILC and 6078 with IDC enrolled in FLEX registry. FLEX (NCT03053193) is a prospective, observational trial that includes stage I-III breast cancer patients who undergo MammaPrint testing (with or without BluePrint) as standard of care, and consent to full transcriptome and clinical data collection. MP High Risk tumors were further stratified into High 1 and High 2. ImPrint and Reprint results were used to determine RPS. A two-tailed proportional z-test was used to assess differences between ILC and IDC as well as between RPS. Results: ILC patients had a significantly lower percentage of MP High Risk tumors in comparison to IDC. Among MP High Risk tumors, those with ILC had significantly more MP High 1 than patients with IDC. BP subtyping in ILC tumors showed significantly lower percentage of Basal, and Luminal B, and higher percentage of Luminal A compared to IDC tumors. For RPS, a higher percentage of ILC tumors were ImPrint-/RePrint- compared to IDC, whereas lower frequencies of ImPrint-/RePrint+ and ImPrint+ were found. Within the ILC ImPrint+ subgroup (n=14), 1 (7.1%) was classified as High 2 and 10 (71.4%) as High 1 and 3 (21.4%) as Low Risk. All 14 (100%) ImPrint+ patients were BP Luminal. In contrast, within the IDC MP High Risk ImPrint+ subgroup (n=328), 233 (71.0%) were High 2, 88 (26.8%) High 1 and 7 Low Risk (2.1%). Furthermore 193 (60.7%) were classified as BP Basal, 2 HER2 (0.6%) and 123 (38.7%) Luminal within this IDC RPS. Conclusions: This is the first study to investigate the distribution of Response Predictive Subtypes in ILC, which will be beneficial to optimize the treatment selection for patients with early-stage HR+/HER2- ILC. Though the percentage of ImPrint+ is lower in ILC, this study revealed a small subset of patients in ILC with potential response to Immunotherapy. Furthermore, these results underscore the heterogeneity of ILC tumors and generate further hypotheses to investigate the immune cell abundance in ILC compared to IDC and correlate immune cell abundance to ImPrint status. Table: Genomic and Tumor Characteristics of HR+HER2- ILC and IDC tumors Citation Format: Rita Mukhtar, Christina Yau, Denise Wolf, Adam Brufsky, Hannah Linden, Natasha Hunter, Reshma Mahtani, Abirami Sivapiragasam, Trevor Feinstein, Fengting Yan, Ian Grady, Priscilla McAuliffe, Michaela Tsai, Joyce O'Shaughnessy, Blanche Mavromatis, Sasha Davis, Josien Haan, William Audeh, Lavanya Samraj, FLEX Investigators' Group. Distribution of MammaPrint, BluePrint, and Response Predictive Subtypes based on ImPrint and Reprint in ER+/HER2- Invasive Lobular Carcinoma – A FLEX sub study [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 PO4-02-03.

Abstract PO1-01-05: Spatial PK/PD Model with HER2 Expression for Predicting Individual Tumor Response to T-DM1

Abstract Introduction: As new antibody drug conjugates are developed, the question of which specific patients will respond to these therapies remains. This work develops a spatial PK/PD model for the antibody drug conjugate trastuzumab emtansine, T-DM1, and explores the relationship between HER2 (target) expression and other individual features of the tumor microenvironment using a computational approach. Methods: We developed a spatial PK/PD model for T-DM1 based upon an allometrically-scaled mouse model (Jumbe, Nelson L., et al., 2010) and the reported outcomes from the KRISTINE trial, which investigated T-DM1 plus pertuzumab in the neoadjuvant early-stage breast cancer setting (Hurvitz, Sara A., et al., 2018). The T-DM1 spatial PK/PD model was then integrated into our previously described 4D biophysical simulation model (Howard, Frederick M., et al., 2022). The biophysical model leverages baseline breast MRI and clinicopathologic features and predicts the individual response to a treatment regimen. Using digital twin tumors from our TumorBank to simulate the KRISTINE trial cohort (n=40), the PD model was parameterized to achieve the trial response rate. To orthogonally validate the spatial PK/PD model, volumetric response and pCR were predicted in an independent cohort of ISPY2 trial patients (n=44) who received T-DM1 followed by dose-dense doxorubicin and cyclophosphamide. We then assessed the added value of the target expression data by developing logistic regression models to predict which patients would achieve pCR based upon HER2 expression alone or HER2 expression combined with the final tumor volume simulated using the spatial PK/PD model. Results: A logistic regression model based upon HER2 expression from transcriptomic data is a strong predictor of response to T-DM1 with an odds ratio of 9.6 and an area under the ROC curve (AUC) of 0.81. The tumorHER2 model, which combines the HER2 expression with the spatial PK/PD model, improves the predictive value of the model with an odds ratio of 34.0 and an AUC of 0.88. Conclusion: The spatial PK/PD improves prognostic predictions compared to HER2 gene expression data alone. In leveraging the SBS biophysical simulation platform, the spatial PK/PD model captures the individual variability in tumor growth rate, tumor perfusion, and drug disposition, all of which influence the treatment efficacy. The rate of target expression will highly influence response to an ADC, but the biophysical model demonstrates that features of the spatial tumor microenvironment also influence response. Table 1. Performance comparison between the SBS tumorHER2model and SBSHER2 Signature. Citation Format: Nicole Hobbs, Bradley Feiger, John Cole, Tricia Carrigan, Joseph Peterson, John Pfeiffer, Daniel Cook. Spatial PK/PD Model with HER2 Expression for Predicting Individual Tumor Response to T-DM1 [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 PO1-01-05.

Abstract 7515: Enhancing immune checkpoint inhibitor efficacy with fulvestrant in estrogen receptor-positive breast cancer

Abstract Introduction: Breast cancer (BC) recurs decades after initial treatment that consists majority of the annual 43,000 BC deaths. Thus, Immune Checkpoint Inhibitor (ICI) therapy that bolster anti-cancer immunity is expected to improve long-term survival with reduced recurrence. Neoadjuvant ICI revolutionized management of triple negative breast cancer (TNBC); however, this represents only 5-7% of all BC cases, where estrogen receptor (ER)-positive subtype that consists of 70% of all BCs is not indicated due to less tumor-infiltrating lymphocytes (TILs). Using I-SPY2 trial we aim to investigate the potency of ER-alpha gene (ESR1) expression as a predictive biomarker, and whether the inhibition of the ER with fulvestrant enhances the efficacy of ICIs in ER+ BC. Methods:Total of 1157 BC patients from two independent cohorts (ISPY2 trial; n= 73, TCGA; n = 1084) were included, encompassing both clinical and transcriptomic profiles. The TIL score was determined using the xCell algorithm. Results:Using the transcriptome of the ISPY2 trial, we found that compared to ESR1 low expression patients, ESR1 high patients were associated with significantly lower pCR rates after neoadjuvant immunotherapy with ICI in whole cohort (n=71, pCR rate=30%, p&amp;lt;0.05) and HR+ subtype (n=50, pCR rate=20%, p&amp;lt;0.001), but not in triple-negative breast cancer (TNBC). ESR1 expression outperformed PD-1, PD-L1, and CTLA4 in Area Under the Curve (AUC) of the whole ISPY2 cohort (AUC=0.75, 0.68, 0.48, and 0.62, respectively), and further pronounced in HR+ BC (AUC=0.88, 0.73, 0.55, and 0.65, respectively), suggesting the potency of ESR1 expression as a predictive biomarker for ICI in HR+ BC. In agreement, ESR1 expression inversely correlated with the amount of intratumoral TILs consistently in both ISPY2 and TCGA cohorts in HR+ BC, but only in TCGA in whole cohort (all p&amp;lt;0.05). Among the intratumoral TILs, CD8+ T cell and M1 Macrophages were found to be less infiltrated in HR+ BC in TCGA. Given that ESR1 expression was associated with less intratumoral TILs and a poor response to ICI, we hypothesized that suppression of ER-alpha would enhance the effectiveness of ICI in HR+ BC in vitro. ER-alpha positive MCF7 cells were incubated with activated peripheral blood mononuclear cells with 1:1 ratio, and treated with 0.1 microgram of anti-PDL1, and 1nM of ER antagonist, fulvestrant, which demonstrated minimum cell killing. We found that ICI therapy with anti-PDL1 significantly decreased the cell viability of the MCF7 cells when ER-alpha expression was suppressed by fulvestrant, while activated peripheral blood mononuclear cells or anti-PDL1 alone or the combination did not exhibit any significant cell killing. Conclusion: We found that ESR1 expression is a predictive biomarker of ICI, and ER antagonist fulvestrant enhances the response of HR+ BC to ICI, suggesting that this combination could expand ICI indications to HR+ BC. Citation Format: Jun Arima, Kohei Chida, Masanori Oshi, Arya Mariam Roy, Kohei Taniguchi, Sang-Woong Lee, Kazuaki Takabe. Enhancing immune checkpoint inhibitor efficacy with fulvestrant in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7515.

Abstract 6804: High IL12family levels are associated with pathologic complete response amongst HER2- patients in the neoadjuvant I-SPY2 TRIAL

Abstract Background: Immune contexture of the tumor microenvironment (TME) is associated with response to therapy. We hypothesize that expression of interleukin (IL) family of cytokines which plays essential roles in the activation and differentiation of immune cells, shapes the landscape of therapeutic response in breast cancers. We leverage published gene expression, molecular and clinical data from the I-SPY2 Trial (PMID35623341) - a neoadjuvant trial for locally advanced breast cancers. We explored the contribution of expression-based signatures of IL signaling at pre-treatment in relation to pathological complete response (pCR). Methods: We curated 32 IL gene sets from literature. Signatures scores characterizing IL signaling were calculated from gene expression profiles of pre-treatment tumor samples from 987 I-SPY2 patients in the first 10 treatment arms of the trial. Association of IL signatures with pCR in HR/HER2 receptor subtypes, and the five I-SPY response-predictive subtypes (RPS) were evaluated using non-parametric tests. Significance levels were defined at adj. p &amp;lt; 0.05. Correlations of IL signatures with multiplex immunofluorescence staining of immune infiltrates in the PD1-inhibitor (PD1i) arm were also assessed. Results: High expression of IL signatures in HER2- patients, regardless of HR status, was associated with pCR especially in the Trebananib, Veliparib/Carboplatin (VC) and PD1i arms. In the PD1i arm, expression of almost all IL signatures was highly correlated with density of T, CD8+ cytotoxic T, CD8- T, and B cells and their colocalization with tumor cells; IL-6 signatures correlated with density of proliferating tumor cells.Within RPS subtypes, enrichment of IL signatures in HER2-/Immune+ such as pro-inflammatory IL-1 and IL-12 signaling was associated with pCR in the VC, PD1i, Ganetespib, and control arms. Furthermore, we identified an IL-12 signature in the HER2-/Immune-/DRD- subtype associated with pCR, with responders characterized by high IL-12 family signaling (Fisher adj. p &amp;lt; 0.05). IL-12 family signaling was positively associated with CD8+ cytotoxic T, Th1 and B cell enrichment, IFN-γ signaling, and inflammatory response overall and in HER2- breast cancers. Conclusion: High pre-treatment expression of IL signatures was associated with HER2- receptor and RPS HER2-/Immune+ subtype-specific response to I-SPY2 agents, and the colocalization of immune and tumor cells in the tumor bed. In HER2- breast cancers, high levels of IL-12 family signaling were associated with response to therapy and enrichment of known IL-12-related pathways that stimulate effective anti-cancer response including CD8+ T cell expansion, Th1 cell differentiation, B cell activation, IFN-γ secretion, and inflammatory response. These findings suggest that IL signaling may serve as a valuable predictive biomarker in this patient population. Citation Format: Kingsley V. Chow, Silver Al Khafaji, Denise Wolf, Christina Yau, Kailey Dubinsky, Elene Tsopurashvili, Kirthiga Ramalingam, Fluorentine van Nouhuijs, Zheyun Xun, Anna van Montfort, Diane Heditsian, Douglas Yee, Laura Esserman, Michael Campbell, Laura van't Veer, Rosalyn W. Sayaman. High IL12family levels are associated with pathologic complete response amongst HER2- patients in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6804.