Subtypes Of Metastatic Breast Cancer Research Articles

Abstract B012: Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive and highly metastatic breast cancer subtype with limited effective treatment options. Immunotherapy with immune checkpoint inhibitors (ICIs) targeting checkpoint molecules have been widely used and have shown therapeutic efficacy in treating multiple malignancies in the last decade. In TNBC, ICI targeting PD-1, alone or in combination with chemotherapy has been approved for the treatment of patients with both early and advanced stage of the disease. Although, there has been significant clinical benefit achieved from anti-PD-1 and chemotherapy, treatment efficacy is often limited to a subgroup of patients. The limited response to ICIs has been attributed to the increased infiltration of immunosuppressive cells (including MDSCs, neutrophils and tumor-associated macrophages), more importantly, the upregulation of alternative immune checkpoint receptors/ligands that may further suppress cytotoxic T cells (CD8+ T cells) in the tumor microenvironment (TME). Therefore, it has been suggested that targeting other immune checkpoint molecules may be more effective in bolstering anti-tumor immune response. V- domain Ig suppressor of T-cell activation (VISTA) is one of the immune checkpoint molecules attracting increasing attention in the context of anti-tumor immunity due to its broad expression across various cancer types and increased expression upon development of immune checkpoint resistance. However, the role of VISTA in regulating the anti-tumor immune response and its therapeutic potential in TNBC remains unclear. In our study, we observed that VISTA is expressed in TNBC patients, predominantly in neutrophils and macrophages. In the Py230 murine breast cancer model, VISTA expression increased upon paclitaxel (PTX), anti-PD-L1 and paclitaxel combined with anti-PD-L1 treatment compared to the control group, suggesting its potential role as a resistance mechanism in sustaining or further inhibiting T cells. In the Py230 model, VISTA blockade increased the infiltration and activation of CD8+ T cells, as evidenced by the higher number of CD8+ T cells, increased CD69+CD8+ T cells, and Granzyme B production, indicating that VISTA blockade improved CD8+ T cells mediated anti-tumor immunity. To functionally test the role of VISTA, we stimulated CD8+ T cells and co-cultured them with naïve macrophages or tumor educated macrophages in the presence or absence of VISTA blocking antibody. Tumor educated macrophages showed increased immunosuppressive activity which was abrogated with VISTA blockade. Moreover, we found that the immunosuppressive effect of macrophages on T cell activation needs direct cell-cell interaction between VISTA on macrophages and T cells. In conclusion, this study shows the mechanism of action of VISTA in the regulation of T cells mediated anti-tumor immune response and provides the rationale for targeting VISTA in TNBC. Citation Format: Maidinaimu Abudula, Yuliana Astuti, Meirion Raymant Raymant, Vijay Sharma, Michael Schmid, Ainhoa Mielgo. Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B012.

349MO Results from a phase Ia/Ib Study of ESG401, a novel Trop2 antibody-drug conjugate, in patients with different subtypes of metastatic breast cancer

349MO Results from a phase Ia/Ib Study of ESG401, a novel Trop2 antibody-drug conjugate, in patients with different subtypes of metastatic breast cancer

Identification of peripheral blood test parameters predicting the response to palbociclib and endocrine therapy for metastatic breast cancer: a retrospective study in a single institution.

Cyclin-dependent kinase 4/6 inhibitors have been used in endocrine therapy for patients with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. Although randomized trials have shown that combined therapies prolong progression-free survival (PFS) in comparison to endocrine monotherapy, the predictors of efficacy are unknown. This study aimed to identify the blood test parameters to predict the effects of palbociclib and endocrine therapy. Seventy-nine patients treated with palbociclib and endocrine therapy between December 2017 and June 2022 were reviewed. We assessed PFS in patients according to factors evaluated based on patient characteristics and peripheral blood tests. Patients in the C-reactive protein (CRP)-high, lactate dehydrogenase (LDH)-high, and albumin (Alb)-low groups had significantly shorter PFS than those in the normal group. A multivariate analysis revealed that high LDH and low Alb levels were independent factors that affected PFS. The Alb-low group had an inferior disease control rate. Patients in the CRP-high, LDH-high, and Alb-low groups who received these therapies as first- or second-line treatments showed poor PFS. Several predictors of the efficacy of palbociclib and endocrine therapy were identified in the peripheral blood test parameters of patients with ER-positive and HER2-negative subtypes of metastatic breast cancer.

4CAST: A phase 1b dose exploration, open-label, single-center study evaluating the safety of INO-464 in combination with chemotherapy in patients with metastatic breast cancer.

e13112 Background: Metastatic triple negative breast cancer (TNBC) has a poor prognosis and the development of new targeted therapies is an area of unmet need. The androgen receptor (AR) antagonist, INO-464, but not abiraterone or enzalutamide, blocks chemotherapy induced cell plasticity to inhibit and primary and metastatic breast cancer (MBC) growth. Single agent INO-464 at a dose of 450mg/d is well tolerated but has low efficacy. Laboratory data demonstrates suppression of metastatic TNBC and an increase in survival with the combination of INO-464 and taxane chemotherapy. Methods: We designed a Phase 1b open-label, single center dose exploration (Part 1) and dose expansion (Part 2) clinical trial of INO-464 450mg/d with docetaxel (IV 75mg/m2 q3wkly) or nab-paclitaxel (IV 100mg/m2 D1/8/15 q4wkly) in any subtype of MBC. The primary objective was to assess the safety and tolerability of INO-464 (450mg/d) and docetaxel or nab-paclitaxel utilizing a rolling 6 design, as well as to determine the maximum tolerated dose (MTD) and the recommended dose of INO-464 to be given with a taxane in the dose expansion study. Secondary objectives included anti-tumor activity and testosterone levels. Results: Recruitment began in August 2022. The cut-off date for this analysis was 19 January 2024. Eight patients have been treated to date and 6 patients were DLT evaluable. All were women with MBC with mean age of 56 (range 48-64) years. Patients had received a median of 5 (range 2 to 7) lines of chemotherapy for MBC. Patient 001 had G3 anaphylaxis due to docetaxel that resolved with adrenaline but came off study and was not DLT evaluable. The protocol was amended to give INO-464 with 1mg of dexamethasone. No DLTs were subsequently observed. Adverse events (AEs) were mostly low grade and chemotherapy related although G2 pneumonitis occurred beyond the DLT period in two patients, one of whom had prior sacituzimab govotecan-related pneumonitis. INO-464-related AEs were G1 fatigue/headache in 4/6 patients. Seven of seven patients had a best response (RECIST1.1) SD, and CA15.3 stability was seen in 4/7 patients. Baseline serum testosterone was detectable in 4/6 patients (range 0.5-1.3 nmol/L, lower limit of detection of <0.5), and remained undetectable or at the lower limit of detection throughout treatment. Conclusions: The MTD of INO-464 is 450mg daily with 0.5mg dexamethasone in combination with docetaxel or nab-paclitaxel chemotherapy. The combination is well tolerated with early efficacy signals. Dose expansion will test the combination in metastatic TNBC with nuclear AR positive breast cancer. Clinical trial information: NCT04947189 .

Differential genomic profiling of the progesterone receptor (PR) negative and estrogen receptor (ER) low metastatic breast cancer subtypes through circulating tumor DNA: A TNBC-like disease subtype?

1039 Background: ER positive/HER2 negative metastatic breast cancer (MBC) is typically regarded as a single entity, although significant clinical differences are often observed, especially in the ER low subgroup (ER<10% regardless of PR status). This study aims to investigate genomic differences among HER2 negative subtypes (i.e., ERlow, PRpos [ER >10%/PRpositve], PRneg [ER >10%/PRnegative], triple negative [TNBC]) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS). Methods: The study analyzed a retrospective cohort of 879 patients (pts) with HER2 negative MBC with ctDNA testing using the Guardant360 NGS panel within a large multicenter academic consortium. ER, PR and HER2 status was defined based on the most updated biopsy. Associations across single nucleotide and copy number variations (SNVs and CNVs) and ER/PR status were tested by multinomial logistic regression in terms of Relative Risk Ratio (RRR), adjusting for significant clinical characteristics (i.e., lines of treatment, metastatic sites, histology). Oncogenic pathway was defined based on reference (1). Prognosis was analyzed through Cox regression for overall survival (OS) defined from time of ctDNA collection. Results: Among the 879 analyzed pts, PRpos was the most prevalent subtype (N:420, 48%), followed by PRneg (N:216, 24%), TNBC (N:209, 24%), and ERlow (N:34, 4%). Compared to PRpos, lobular MBC was less common in TNBC (RRR: 0.33, P=0.001), while lung involvement was more frequent in TNBC (RRR: 1.53, P=0.019), and liver involvement in PRneg (RRR: 1.44, P=0.035). In contrast, bone metastases were less common in TNBC and ERlow (RRR: 0.26, P<0.001 and RRR: 0.45, P=0.03, respectively). After multivariable analysis, ERlow and TNBC were associated with a higher incidence of TP53 SNVs (RRR: 5.42, P<0.001 and RRR: 3.08, P=0.007, respectively) and a lower incidence of ESR1 SNVs (RRR: 0.23, P=0.025 and not detected, respectively). TNBC exhibited significantly fewer PIK3CA SNVs (RRR: 0.41, P=0.005) and CCND1 CNVs (RRR: 0.20, P=0.009), along with a higher frequency of CCNE1 CNVs (RRR: 6.47, P<0.001). PRneg had a lower incidence of ESR1 SNVs (RRR: 0.52, P=0.013) and more frequent FGFR1 CNVs (RRR: 2.61, P=0.004). Similar results were observed across oncogenic pathways. Compared to PRpos, OS analysis indicated poorer outcomes across all subgroups (PRneg HR: 1.29 P = 0.035, TNBC HR: 1.96 P<0.001, ERlow HR: 2.32 P<0.001). Conclusions: This study highlights several clinical and molecular similarities between TNBC and ERlow MBC, while suggesting specific endocrine resistance mechanisms in PRneg that are less reliant on ESR1 and more exposed to alternative mechanisms such as FGFR1 amplification. These findings underscore the importance of more precise disease subtyping to inform future drug development trials. 1. Sanchez-Vega F et al, Cell. 2018.

Abstract PO2-05-14: Prognosis and clinicopathological characteristics of HER2-negative metastatic breast cancers with low expression of hormone receptors: real world evidence from the French ESME cohort

Abstract Background: HER2-negative breast cancers expressing low (1-9 %) immunohistochemistry levels of hormone receptors (HR, estrogen and progesterone receptors), remain an uncertain category, being considered either as triple negative breast cancers (TNBC) or HR-positive (HR+) tumors across guidelines or approvals. The issues regarding this clinical subgroup are both prognostic, with the need of a better definition of their clinical outcome, as well as predictive. Methods: The ESME database is a French National cohort of all consecutive patients who initiated a first-line treatment for metastatic breast cancer (MBC) from 2008 on, in one of the 18 French Comprehensive Cancer Centers. Patients with HER2-negative (0 to 2+, ISH negative) MBC and known estrogen receptor (ER) and progesterone receptors (PR) expression levels were selected. Primary objective was to evaluate crude overall survival (OS) in HER2-/HR-low (ER and PR 0-9 and not TNBC) MBC pts compared with those with TNBC and HR+ disease defined by ER and PR < 1%, and ER ± PR ≥ 10%, respectively. In these 3 populations, predefined secondary objectives were to evaluate PFS under first line chemotherapy (CT), to describe clinical characteristics and the distribution of genetic abnormalities identified at the time of MBC diagnosis. Results: Out of 30,459 patients in the ESME database initiating their MBC treatment between 01/2008 and 01/2021, 19109 patients (2113 TNBC; 228 HR-low; 16768 HR+) were eligible for this analysis. Median follow-up was 58.0 months (95%CI [56.6; 59.0]). Median OS were 15.7 months [15.0-16.8], 19.1 months [15.5-22.4] and 44.6 months [43.8-45.5] in the TNBC, HR-low and HR+ subgroups, respectively. The multivariable analysis adjusted on age at MBC diagnosis, pathological grade and subtype, metastatic-free interval, number and sites of metastases and HER2 status (0 vs. 1-2+), identified no significant OS difference between HR-low and TNBC subgroups (HR=0.95, 95%CI 0.79-1.14), while HR+ patients had a better OS than TNBC patients (reference) (HR=0.50, 95%CI 0.47-0.53). Of these 19109 pts, 8910 received first-line CT regimen (1960 TNBC; 195 HR-low; 6755 HR+): taxanes-based: 66.7%, anthracyclines-based: 32.9%, capecitabine: 18%. Median PFS under first line CT were 5.3 (5.1-5.6), 5.1 (4.1-6.2) and 10.2 months (9.9-10.6) for TNBC, HR-low and HR+ subgroups, respectively. In the multivariable analysis adjusted on the same variables, the HR-low subgroup had a statistically poorer PFS compared to TNBC (HR=1.24, 95% CI 1.04-1.49). On the other hand, the HR+ subgroup had a better PFS than the TNBC (HR=0.74, 95%CI [0.70; 0.78]). Baseline germline BRCA1 mutations (at MBC diagnosis) were present in 4.5% of the TNBC cases, 2.6% of the HR-low cases, and 0.4% of the HR+ cases. The distribution of germline BRCA2 mutations was more homogeneous between the three groups (0.9% of TNBC cases, 1.3% of HR-low cases and 1.1 % of HR+ cases). Conclusions: In this large cohort, patients with HER2-negative, HR-low MBC seem to share similar general prognosis, oncogenetic features and PFS under standard chemotherapy treatment with TNBC patients. Citation Format: Marie Alexandre, Ludovic Gauthier, Audrey Mailliez, Thibault DE LA MOTTE ROUGE, Thomas Bachelot, Monica Arnedos, Florence Dalenc, Etienne Brain, Jean-Marc Ferrero, Vincent Massard, Isabelle Desmoulins, Marie-Ange Mouret-Reynier, Christelle Levy, Anthony Gonçalves, Marianne Leheurteur, Thierry Petit, Jean-Sébastien Frenel, Lise Bosquet, Suzette Delaloge, William Jacot. Prognosis and clinicopathological characteristics of HER2-negative metastatic breast cancers with low expression of hormone receptors: real world evidence from the French ESME 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 PO2-05-14.

Genomic spectrum of actionable alterations in serial cell free DNA (cfDNA) analysis of patients with metastatic breast cancer

We aimed to study the incidence and genomic spectrum of actionable alterations (AA) detected in serial cfDNA collections from patients with metastatic breast cancer (MBC). Patients with MBC who underwent plasma-based cfDNA testing (Guardant360®) between 2015 and 2021 at an academic institution were included. For patients with serial draws, new pathogenic alterations in each draw were classified as actionable alterations (AA) if they met ESCAT I or II criteria of the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). A total of 344 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) MBC, 95 patients with triple-negative (TN) MBC and 42 patients with HER2-positive (HER2 + ) MBC had a baseline (BL) cfDNA draw. Of these, 139 HR+/HER2-, 33 TN and 13 HER2+ patients underwent subsequent cfDNA draws. In the HR+/HER2- cohort, the proportion of patients with new AA decreased from 63% at BL to 27–33% in the 2nd-4th draws (p < 0.0001). While some of the new AA in subsequent draws from patients with HR+/HER2- MBC were new actionable variants in the same genes that were known to be altered in previous draws, 10-24% of patients had new AA in previously unaltered genes. The incidence of new AA also decreased with subsequent draws in the TN and HER2+ cohorts (TN: 25% to 0–9%, HER2 + : 38% to 14–15%). While the incidence of new AA in serial cfDNA decreased with subsequent draws across all MBC subtypes, new alterations with a potential impact on treatment selection continued to emerge, particularly for patients with HR+/HER2- MBC.

Gene expression in metastatic breast cancer-patterns in primary tumors and metastatic tissue with prognostic potential.

Background: Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes. Methods: RNA was extracted from the PT, lymph node metastasis (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity. Results: Compared to DM GEX, GEX of the PT was at least equally useful for predicting MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort. Conclusion: GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Metixene is an incomplete autophagy inducer in preclinical models of metastatic cancer and brain metastases.

A paucity of chemotherapeutic options for metastatic brain cancer limits patient survival and portends poor clinical outcomes. Using a CNS small-molecule inhibitor library of 320 agents known to be blood-brain barrier permeable and approved by the FDA, we interrogated breast cancer brain metastasis vulnerabilities to identify an effective agent. Metixene, an antiparkinsonian drug, was identified as a top therapeutic agent that was capable of decreasing cellular viability and inducing cell death across different metastatic breast cancer subtypes. This agent significantly reduced mammary tumor size in orthotopic xenograft assays and improved survival in an intracardiac model of multiorgan site metastases. Metixene further extended survival in mice bearing intracranial xenografts and in an intracarotid mouse model of multiple brain metastases. Functional analysis revealed that metixene induced incomplete autophagy through N-Myc downstream regulated 1 (NDRG1) phosphorylation, thereby leading to caspase-mediated apoptosis in both primary and brain-metastatic cells, regardless of cancer subtype or origin. CRISPR/Cas9 KO of NDRG1 led to autophagy completion and reversal of the metixene apoptotic effect. Metixene is a promising therapeutic agent against metastatic brain cancer, with minimal reported side effects in humans, which merits consideration for clinical translation.

A transcriptome-informed QSP model of metastatic triple-negative breast cancer identifies predictive biomarkers for PD-1 inhibition.

Triple-negative breast cancer (TNBC), a highly metastatic breast cancer subtype, has limited treatment options. While a small number of patients attain clinical benefit with single-agent checkpoint inhibitors, identifying these patients before the therapy remains challenging. Here, we developed a transcriptome-informed quantitative systems pharmacology model of metastatic TNBC by integrating heterogenous metastatic tumors. In silico clinical trial with an anti-PD-1 drug, pembrolizumab, predicted that several features, such as the density of antigen-presenting cells, the fraction of cytotoxic T cells in lymph nodes, and the richness of cancer clones in tumors, could serve individually as biomarkers but had a higher predictive power as combinations of two biomarkers. We showed that PD-1 inhibition neither consistently enhanced all antitumorigenic factors nor suppressed all protumorigenic factors but ultimately reduced the tumor carrying capacity. Collectively, our predictions suggest several candidate biomarkers that might effectively predict the response to pembrolizumab monotherapy and potential therapeutic targets to develop treatment strategies for metastatic TNBC.

Comparison of real-world outcomes over time in a large population-based cohort of patients with metastatic breast cancer.

e13112 Background: The treatment of metastatic breast cancer (MBC) has evolved over the past two decades, with a number of new therapeutics being introduced. Real-world evidence is needed to evaluate whether these new therapeutics have made an impact on population survival, as improvements in survival seen within clinical trials do not necessarily translate into the real world. The purpose of this study was to investigate whether survival for each subtype of MBC has improved in patients diagnosed with MBC between 2003 and 2017. In addition, for the HER2+ subtype, we aimed to characterize and correlate changes in the use of anti-HER2 therapeutics with any observed real-world survival changes. Methods: The study included residents of British Columbia, Canada who were diagnosed with de novo or recurrent MBC during one of the following four time periods: 2003 to 2005, 2007 to 2009, 2011 to 2013, and 2014 to 2017. Patients were included whether or not they received any systemic therapies for MBC. Patient, disease, and treatment characteristics were obtained through medical records and the Breast Cancer Outcomes Unit database. Survival estimates were calculated using the Kaplan-Meier method. Univariable analyses were performed using the log-rank test, and multivariable analyses (MVA) were performed using the Cox proportional hazards model. Results: 5,924 patients were diagnosed with MBC during one of the four time periods. 3,756 (63%) had hormone receptor positive (HR+) subtype, 1,145 (19%) had HER2+ subtype, 770 (13%) had triple negative (TN) subtype, and 253 (4%) had unknown subtype. For the HR+ subtype, overall survival (OS) was similar for cohorts 1 to 3 (median survivals: 751 days, 704 days, and 702 days). However, OS was longer for cohort 4 (median survival: 855 days) compared to cohort 3 ( p &lt; 0.001). For the HER2+ subtype, OS was similar for cohorts 1 to 3 (median survivals: 594 days, 511 days, and 592 days). However, OS was longer for cohort 4 (median survival: 791 days) compared to cohort 3 ( p = 0.03). For the TN subtype, there was no significant change in OS for the four cohorts (median survivals: 234 days, 236 days, 274 days, and 255 days). On MVA, younger age, non-TN subtype, time cohort 4, de novo presentation, and disease-free interval of &gt; 12 months were associated with improved OS. For the HER2+ subtype, a greater percentage of patients in the later cohorts were treated with pertuzumab ( p &lt; 0.001) and trastuzumab emtansine (T-DM1) ( p &lt; 0.001), with 12.4% and 36.3% respectively receiving these drugs in cohort 3 compared to 80.9% and 52.5% respectively in cohort 4. Conclusions: Patients diagnosed with MBC of the HR+ or HER2+ subtypes in the 2014 to 2017 time period had improved real-world outcomes compared to those diagnosed in earlier time periods. Improvements in real-world outcomes were likely associated with new therapeutic agents with demonstrated OS benefit in trial settings.

Distal inflammatory priming of normal mammary tissue by the gut microbiome drives breast tumor metastasis via metabolic dysregulation

Abstract Hormone receptor-positive (HR +), HER2 −is the most prevalent metastatic breast cancer subtype, constituting 73.1% of the metastatic disease population in the US. Despite targeted therapies that have increased long-term survival, many patients develop and eventually succumb to metastatic disease. Tumor dissemination occurs early during disease progression and is driven by cellular and molecular changes in the tissue. Though host-intrinsic factors that preferentially predispose certain individuals to metastatic disease are poorly defined, gut microbial health has become increasingly recognized as a determinant of the metastatic potential of breast tumors. We demonstrated that commensal dysbiosis, an inflammatory gut microbiome with low biodiversity, drives HR +tumor metastasis. Our work identified that a dysbiosis-induced CCL2/mast cell axis in normal (non-tumor-bearing) mammary tissues acts as a master regulator of early metastasis. Our present goal is to define how gut dysbiosis triggers tissue inflammation. We present evidence that dysbiosis-induced metabolic dysregulation increases early dissemination of HR +tumors by initiating cellular and molecular changes in normal mammary tissue. Dysbiosis results in systemic insulin-glucose dynamics that resemble an insulin resistant phenotype. Additionally, we find that acylcarnitine species, byproducts of incomplete fatty acid β-oxidation which we hypothesize promote local tissue inflammation, accumulate in normal mammary tissues of dysbiotic mice. Altogether, we propose that commensal dysbiosis triggers a systemic metabolic shift that enhances the metastatic potential of HR +tumors by shaping the immune landscape of the pre-cancerous mammary tissues. Supported by grants from NIH (1RO1 CA253285, 2T32AI007496-26A1)

Abstract P1-05-12: Evaluation of the benefit of the tumor marker cancer antigen 15–3 (CA 15-3) in different subtypes of metastatic breast cancer

Abstract Background: Cancer antigen 15-3 (CA 15-3) is the most commonly used tumor marker in patients with breast cancer (BC). However, the usefulness of CA 15-3 is still controversial and there is lack of evidence for its role in metastatic BC. We performed a retrospective analysis of the value of CA 15-3 for monitoring and diagnosis in subgroups of metastatic BC. Method: This retrospective study included three cohorts of a total of 196 patients with advanced or metastatic BC treated during the years 2015-2020. Patients’ data, such as subtype, hormone receptor status, and CA 15-3 values, were retrieved from a treatment database to find out how well the CA 15-3 levels followed the disease progression or remission over time. An increase or decrease of CA 15-3 less than 10% indicated stable disease and changes larger than 10% indicated progress or response of therapy. The patients were then categorized into one of six groups based on the proportion of correct CA 15-3 values in each patient during the course of the disease. A proportion of correct values of 95% or higher placed the patient in category 5, between 90% and 95% category 4, 75% to 90% were category 3, 50% to 75% category 2 and below 50% category 1. Patients without any elevated CA 15-3 values and with no evidence of association with a trend of CA 15-3 and disease progression were categorized as 0. Patients in categories 3, 4, and 5 were classified with approved CA 15-3 conformity and categories 4 and 5 with excellent conformity. Results: The median number of samples per patient was 27 (range 5-102) and at diagnosis of metastatic disease, 79%, 66% and 60% of patients had elevated CA 15-3 in the luminal, HER2-positive, and TNBC groups, respectively. Overall, 72.4%, 95% confidence intervals (CI) (66.2-78.7) of the patients had an approved conformity of CA 15-3 to monitor the course of the disease. The benefit of CA 15-3 differed significantly between the subtypes, luminal BC had an approved conformity for 94%, 95% CI (88.6-99.1) of the patients, HER2 BC 63%, 95% CI (52.1-73.5) and for TNBC 46% (95% CI 29.9-62.0) of patients. In patients with Luminal A, the conformity was excellent in 84% of patients compared to 60% of patients with Luminal B. Estrogen receptor status (ER) was the single most important factor for the value of CA 15-3, with an approved conformity of 91%, 95% CI (85.6-95.7) of ER-positive patients and 37%, 95% CI (25.3-48.2) of all ER-negative patients. Elevated baseline levels of CA 15-3 in the diagnosis of metastatic BC increased the benefit of the marker to approved levels in 62.5% of the ER-negative patients, but ER-positive patients mostly had approved conformity, despite negative CA 15-3 at baseline. Visceral or bone metastases further improved the utility of CA 15-3 and soft tissue or solitary CNS metastases decreased it. Conclusion: The benefit of serum marker CA 15-3 in monitoring patients with metastatic BC depends mainly on whether the tumor expresses ER or not. ER-positive patients were excellently monitored in most cases and elevated CA 15-3 values at baseline indicated potential utility in ER-negative patients. Citation Format: Henrik Lindman, Ebba Cederqvist Molin, Martin Jernling, Aglia Schiza. Evaluation of the benefit of the tumor marker cancer antigen 15–3 (CA 15-3) in different subtypes of metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-12.

Abstract P2-20-03: Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model

Abstract Background: Intrinsic resistance to immunotherapy observed in breast cancer is attributed to low neoantigen levels, defective antigen presentation, low mutational burden, reduced programmed death ligand (PD-L1) expression and the presence of immunosuppressive signals like transforming growth factor-beta (TGFβ) in the tumor microenvironment (TME). These collectively attenuate the effector functions of T cells and natural killer cells (NK cells). NK cells are an important component of innate immunity. While generally TGFβ dampens the NK cell response in the breast cancer TME, chronic stimulation in vivo with TGFβ during the expansion process of donor NK cells produces TGFβ imprinted NK cells which exhibit high cytotoxicity and resistance to suppression by TGFβ due to down regulation of SMAD3 and hypersecretion of interferon gamma and tumor necrosis factor alpha. NK cell activation also promotes antibody dependent cellular cytotoxicity (ADCC) and augments monoclonal antibody directed killing. GD2 is a disialoganglioside and a tumor-associated antigen with limited expression in healthy tissues and overexpression in a variety of cancers, including breast cancer. Up to 60% of high grade breast cancers have GD2 expression with higher expression linked to worse overall survival. Given GD2 expression in aggressive BC subtypes and the role of NK cells in ADCC and antigen presentation, there is a strong rationale for evaluating an anti-GD2 antibody along with NK cells in the immunosuppressive breast TME. Based upon established preclinical data, we hypothesized that combining (1) an IgG1 anti-GD2 monoclonal antibody (anti-GD2), (2) TGFβ imprinted, IL-21 expanded allogenic universal donor NK cells (TGFβiNK) and (3) the standard chemotherapy agent gemcitabine (gem) which has immunomodulatory effects including improved NK cell function, will improve tumor control and survival in a TNBC mouse model. Methods: First, we evaluated if anti-GD2 treatment would increase ADCC against MD-MBA-231 tumor cells. To do this, tumor cells with or without anti-GD2 were cocultured with TGFβiNK + anti-GD2 and evaluated by real time cell analysis (RTCA). Next we evaluated the effect of gem on both tumor and immune cells. Finally to evaluate the effect of our novel combination therapy, female NSG mice were injected with 1 × 10e6 MDA-MB-231 cells into the mammary fat on day 0 and tumor burden was followed for 83 days. On day 21, mice were divided into 4 groups: control, gem only (standard of care), TGFβiNK+anti-GD2 (NK-GD2), and gem+TGFβiNK+anti-GD2 (G-NK-GD2) and tumor growth was measured weekly. Mice were observed for toxicity and bioluminescence evaluation for NK cells were performed prior to each cycle. Results: RTCA data confirmed anti-GD2 treatment increased ADCC mediated cell lysis by TGFβiNK. Furthermore, at clinically relevant doses, gem did not alter GD2 expression by flow cytometry on MDA-MB-231 tumor cells but induced MICA expression which suggests potential improved NK cell activity. In the mouse model, monitoring of mouse weights showed greater weight stability in the G-NK-GD2 group, suggesting a tolerable regimen. Serial measurement of tumors showed a significant reduction in tumor growth in NK-GD2 and G-NK-GD2 arms when compared to Gem or control. Additionally, mice who received G-NK-GD2 or NK-GD2 had significantly improved survival. Histological evaluation and immune assessment of the mice are ongoing. Conclusion: Our data showed no significant toxicities in the murine model and improved efficacy of G-NK-GD2 over the standard of care gemcitabine. Our preclinical data supports moving forward with a phase 1b/2 Bench-To-Bedside clinical trial, that will evaluate this combination in patients with aggressive subtypes of metastatic breast cancer where there is a great clinical need for effective treatments. Citation Format: Marcelo Pereira, Prashant Trikha, Sumithira Vasu, Zihai Li, Daniel Stover, Dean Lee, Margaret Gatti-Mays. Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-03.

Abstract PD2-08: Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Currently used neoadjuvant chemotherapy for TNBC, such as taxanes, and/or anthracyclines demonstrate an initial treatment response but is often followed by drug resistance, tumor relapse, and high proclivity to develop metastases. There is an unmet need to develop effective combination therapies against metastatic TNBC (mTNBC). eIF4A1 is an mRNA helicase that help translate many oncogenic mRNAs including PD-L1 (through STAT1 translational regulation) that are involved in tumor growth, chemoresistance and metastases. Our prior studies pointed out that eIF4A1 is intricately linked to cancer stemness and drug resistance in TNBC. Emerging evidence has revealed that the tumor immune microenvironment (TIME) of TNBC do consist of mononuclear cells and lymphocytes. Combining chemotherapy with immune checkpoint inhibition had demonstrated a significant benefit for high-risk TNBC patients. In this study, we examine the efficacy of targeting eIF4A1 along with anti-PD-L1 therapy as an effective therapeutic strategy to limit cancer stemness, primary tumor progression and metastasis in an immunocompetent murine model of TNBC. Approach: Initially, we examined the human TNBC biospecimens and found that eIF4A1 protein level is upregulated in drug-resistant cases. STAT1 is an upstream regulator of PD-L1 and a downstream effector of eIF4A1. The protein level of STAT1 was significantly elevated as assessed by immunoblotting of human tumor lysates. In human TNBC cells, when eIF4A1 is pharmacologically or genetically targeted, PD-L1 was downregulated at the protein level. Based on this premise, we targeted eIF4A1 with the small molecule inhibitor, Rocaglamide A (RocA), along with anti-PD-L1 immunotherapy in orthotopically implanted 4T1 mouse TNBC tumors in BALB/c mice. The treatment arms were 1) Control (vehicle for RocA + control IgG) 2) RocA 3) RocA + anti-PD-L1 antibodies 4) Control IgG only 5) Anti-PD-L1 antibodies only with 10 mice in each group. Results: The combination treatment comprising of RocA and anti-PD-L1 antibodies worked out efficaciously as the primary tumor volume, wet tumor weight, bioluminescent tumor imaging and quantitation were significantly lower than the combination vehicle control. Furthermore, metastasis to the lungs were significantly impaired (1 out of 10 mice only had metastasis and that too it was small). On the contrary, ‘RocA single arm’ group had 6 out of 10 mice with lung metastasis. The lung metastases were observed in 4 out of 10 mice in the PD-L1 single arm and 6 out of 10 mice in control IgG single arm group. The RocA was tolerated well at 0.6 mg/kg with no significant change in the body weight and the liver and kidney function test values were within the normal range. Importantly, the combination treatment with RocA and PD-L1 was also tolerated well. Conclusions: The combination therapy involving RocA and PD-L1 had the significant impact in reducing lung metastases and may pave the way for phase I/II clinical trials. Citation Format: Dharmindra Dulal, Andrew Boring, Dayanidhi Raman. Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-08.

Abstract P5-14-09: Comparison of cell-free DNA genomics of breast cancer associated-chest wall disease vs. age &amp; subtype matched controls with metastatic breast cancer not involving the chest wall

Abstract Background: Breast cancer associated-chest wall disease (CWD) clinically behaves in an aggressive manner. However, little is known about the genomics of CWD. Cell-free DNA (cfDNA) can identify oncogenic mutations in metastatic breast cancer (MBC). We hypothesized that the cfDNA genomics of CWD may vary from MBC cases without CWD. We compared the cfDNA genomics of patients with CWD to that of age and subtype matched MBC controls without CWD. Methods: Patients with MBC at an academic institution who underwent cfDNA testing (Guardant360, next generation sequencing (NGS), 74 gene assay) from 2/2016-2/2021 were identified. Patients with documented CWD (chest wall recurrence with nodules, ulceration, and/or skin metastases on imaging and/or examination) at the time of cfDNA testing (coinciding with MBC diagnosis) were included in the CWD cohort. Age and subtype matched MBC controls without CWD (CON) during the same time period with cfDNA results at MBC diagnosis were identified. A retrospective review was conducted to determine clinical features and cfDNA genomics of CWD and CON. A two-sample test of proportions was used to compare CWD to CON, with p&amp;lt; 0.05 for statistical significance. Results: Thirty-one patients with CWD and 63 CON were identified. Both groups were well-matched in median age at MBC diagnosis (CWD 57 vs. CON 59 years, p=0.93) and subtype distribution (CWD: TNBC 35%, HR+/HER2- 58%, HER2+ 6%; CON: TNBC 29%, HR+/HER2- 65%, HER2+ 6%, p=0.78). Patients also had similar racial distribution in both cohorts (p=0.57). Ninety percent (28/31) of CWD vs. 90% (57/63) of CON had ≥1 mutation detectable in cfDNA (p=0.62). Median number of cfDNA mutations for CWD was 4.5 (range 0-16) vs. 4 (range 0-21) in CON (p=0.32). The most common cfDNA mutations in CWD were TP53 (58%), NOTCH1 (19%), PIK3CA (16%), BRCA1 (13%), NF1 (13%), FGFR2 (13%), ESR1 (13%), STK11 (10%), NTRK1 (10%), APC (10%), and KIT (10%). In comparison, the most common cfDNA mutations in CON were TP53 (54%), PIK3CA (35%), ESR1 (14%), GATA3 (13%), and ATM (11%). Table 1 depicts mutations that varied between CWD and CON which were statistically significant in ≥1 cohort analyzed. In HR+/HER2- MBC, NOTCH1, STK11, NTRK1, DDR2, and NF1 were significantly more common in CWD than CON. For TNBC, NOTCH1 was numerically more common in CWD vs. CON (p=0.06). Conclusions: The cfDNA genomic spectrum of CWD varies from MBC that does not infiltrate the chest wall. Mutations that are associated with metastasis (NOTCH1), inhibition of tumor suppression (STK11), tumor migration (DDR2), proliferation (NTRK1), and endocrine resistance (NF1) were significantly more common in HR+/HER2- CWD than matched controls. Further research is needed to validate these findings and determine the impact of matched targeted therapies for CWD. Table 1 N/A: mutation not observed Citation Format: Neelima Vidula, Lianne Ryan, Andrzej Niemierko, Dejan Juric, Steven J. Isakoff, Francys Verdial, Marjan Azin, Laura A. Petrillo, Beverly Moy, Leif Ellisen, Shadmehr Demehri, Aditya Bardia. Comparison of cell-free DNA genomics of breast cancer associated-chest wall disease vs. age &amp; subtype matched controls with metastatic breast cancer not involving the chest wall [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-09.

Abstract P1-08-07: Educational Opportunities to Improve HER2 Testing and Use of Newer Therapies for HER2-Positive MBC Among Community Healthcare Professionals

Abstract Background: The management of HER2-positive (HER2+) metastatic breast cancer (MBC) has continued to evolve, with multiple FDA approvals of novel HER2-targeted agents since late 2019. Considering the growing HER2+ MBC treatment armamentarium and the emerging HER2-low MBC subtype, we undertook an analysis of the current implementation of guideline and expert recommendations for HER2 testing and treatment of HER2+ MBC with recently approved agents among oncology healthcare professionals (HCPs) participating in educational programs on these topics. Methods: Between January 2020 and April 2022, we conducted several expert-designed educational activities for HCPs focusing on recommended HER2 testing and treatment of HER2+ MBC. Polling questions on HCP knowledge, practice patterns, and confidence in the evaluation of HER2 status and the use of newer targeted therapies in the setting of HER2+ MBC were assessed across the activities and compared with expert recommendations. Results: Among 129 HCPs participating in 2 educational activities held from July 2020 to March 2021, 53% reported confidence to appropriately apply current HER2 testing guidelines to patient care. Similarly, among 407 HCPs who participated in 4 educational programs from May 2020 to January 2021, 24% reported confidence to incorporate recently approved drugs (including trastuzumab deruxtecan [T-DXd], neratinib, and tucatinib) into the care of patients with HER2+ MBC. The percentage of HCPs reporting confidence in the use of these new drugs remained low over this time period. Comparison of treatment recommendations for various HER2+ MBC case scenarios across 7 educational activities from January 2020 through April 2022 showed considerable discordance between experts and HCPs regarding the implementation of newly approved HER2-targeted regimens (Table 1). For patients with disease progression after 2 lines of therapy and no central nervous system (CNS) metastases, 27% to 57% of HCPs (n = 571) chose T-DXd in agreement with experts. For patients with disease progression after 2 lines of therapy with CNS metastases, 27% to 42% of HCPs (n = 597) chose a tucatinib-based regimen in agreement with experts. Treatment choice concurrence between HCPs and experts did not increase over the course of this study for either of these patient scenarios. Conclusions: These data suggest that some HCPs are challenged to optimally incorporate recommendations for HER2 testing and the implementation of novel HER2-targeted therapies in the care of patients with HER2+ MBC. Educational activities designed to improve the knowledge and confidence of HCPs would benefit patients with HER2+ MBC. A detailed analysis of trends over time and by role on the care team will be presented. Table 1. Expert and HCP Treatment Selections for Case Scenarios in HER2+ MBC. Citation Format: Ryan P. Topping, Rachael M. Andrie, Kristen Rosenthal, Timothy A. Quill. Educational Opportunities to Improve HER2 Testing and Use of Newer Therapies for HER2-Positive MBC Among Community Healthcare Professionals [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-08-07.

Treatment Sequencing Patterns and Associated Direct Medical Costs of Metastatic Breast Cancer Care in the United States, 2011 to 2021

Advances in treatment of metastatic breast cancer (MBC) led to changes in clinical practice and treatment costs in the US over the past decade. There is limited information on current MBC treatment sequences and associated costs by MBC subtype in the US. To identify treatment patterns by MBC subtype and associated anticancer and supportive drug costs from health care sector and Medicare perspectives. This economic evaluation analyzed data of patients with MBC obtained from the nationwide Flatiron Health database, an electronic health record-derived, deidentified database with data from community and academic practices across the US from 2011 to 2021. Participants included women aged at least 18 years diagnosed with MBC, who had at least 6 months of follow-up data, known hormone receptor (HR) and human epidermal growth factor receptor 2 (ERBB2) receptor status, and at least 1 documented line of therapy. Patients with documented receipt of clinical study drugs were excluded. Data were analyzed from June 2021 to May 2022. Outcomes of interest were frequency of different drug regimens received as a line of therapy by subtype for the first 5 lines and mean medical costs of documented anticancer treatment and supportive care drugs per patient by MBC subtype and years since metastatic diagnosis, indexed to 2021 US dollars. Among 15 215 patients (10 171 patients [66.85%] with HR-positive and ERBB2-negative MBC; 2785 patients [18.30%] with HR-positive and ERBB2-positive MBC; 802 patients [5.27%] with HR-negative and ERBB2-positive MBC; 1457 patients [9.58%] with triple-negative breast cancer [TNBC]) who met eligibility criteria, 1777 (11.68%) were African American, 363 (2.39%) were Asian, and 9800 (64.41%) were White; the median (range) age was 64 (21-84) years. The mean total per-patient treatment and supportive care drug cost using publicly available Medicare prices was $334 812 for patients with HR-positive and ERBB2-positive MBC, $284 609 for patients with HR-negative and ERBB2-positive MBC, $104 774 for patients with HR-positive and ERBB2-negative MBC, and $54 355 for patients with TNBC. From 2011 to 2019 (most recent complete year 1 data are for patients diagnosed in 2019), annual costs in year 1 increased from $12 986 to $80 563 for ERBB2-negative and HR-positive MBC, $99 997 to $156 712 for ERBB2-positive and HR-positive MBC, and $31 397 to $53 775 for TNBC. This economic evaluation found that drug costs related to MBC treatment increased between 2011 and 2021 and differed by tumor subtype. These findings suggest the growing financial burden of MBC treatment in the US and highlights the importance of performing more accurate cost-effectiveness analysis of novel adjuvant therapies that aim to reduce metastatic recurrence rates for early-stage breast cancer.

Total Lesion Glycolysis Levels as Predictive Indicators in Patients With Metastatic and Recurrent Breast Cancer Undergoing Endocrine Therapy With or Without CDK4/6 Inhibitor.

Endocrine therapy (ET) with or without CDK4/6 inhibitors is the primary treatment choice for patients with estrogen receptor (ER)-positive and HER2-negative subtype of metastatic breast cancer (MBC). We examined the metabolic parameters identified using 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) in terms of sensitivity, since no predictive factors exist. We included 136 patients with MBC treated with ET alone (n=107) or combined with CDK4/6 inhibitor (n=29) and examined using FDG-PET before treatment began. The highest maximum value of the standard uptake value (SUVmax), whole-body metabolic tumor volume (MTV), and total lesion glycolysis (TLG) were calculated. Progression-free survival (PFS) was significantly longer in patients with low levels of MTV, TLG, and SUVmax than those with higher levels (median PFS 49.5 vs. 20.7 months, p=0.001 for MTV, 49.5 vs. 20.7 months, p=0.0016 for TLG, 37.0 vs. 20.7 months, p=0.012 for SUVmax). Multivariable analysis revealed that TLG (hazard ratio=6.383, 95% confidence interval=1.167-34.913, p=0.033) was independently and significantly associated with PFS. The relationship between TLG levels and PFS was significant in patients treated with ET with (p=0.0054) and without (p=0.0188) CDK4/6 inhibitor. TLG at baseline was a significant predictor for sensitivity to ET alone or combined with CDK4/6 inhibitor. These data may be useful to identify patients that would benefit from ET.

Reliability of CA15-3 Tumor Marker in Monitoring Therapeutic Response in Different Molecular Subtypes of Metastatic Breast Cancer

Reliability of CA15-3 Tumor Marker in Monitoring Therapeutic Response in Different Molecular Subtypes of Metastatic Breast Cancer