Triple-negative Breast Cancer Patients Research Articles

Abstract PO5-25-04: Predictive value of neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios in patients with triple negative breast cancer treated with pembrolizumab

Abstract Background: Immune checkpoint inhibitors (ICIs) offer new treatment possibilities for women with triple-negative breast cancer (TNBC) – one of the most challenging breast cancer subtypes. The PD-1 inhibitor pembrolizumab (pembro; Keytruda®) is approved in metastatic TNBC (mTNBC) patients with PD-L1(+) tumors, and for neoadjuvant treatment (NAT) in stage II/III TNBC [1, 2]. Residual cancer burden (RCB) after NAT is highly prognostic, with pathologic complete response (pCR, RCB 0) anticipating long-term survival [3]. In mTNBC, pembro plus chemotherapy in the 1st-line improves progression free survival (PFS), but durable responses are rare. Optimizing ICI use in TNBC thus remains an unmet need, and defining easily assessable markers of response is crucial. Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are peripheral blood surrogates of tumor inflammation and have prognostic and predictive value in TNBC [4-8]. Elevated NLR (≥ 5) and PLR (≥ 200) correlate with worse outcomes upon ICIs in melanoma and non-small-cell lung cancer [9, 11-14]; however, this has not been studied in breast cancer [9, 10]. We evaluated the relationship of baseline NLR and PLR with pembro responses in TNBC patients. Methods: We retrospectively reviewed data from TNBC patients who completed pembro between 10/2/20 - 6/1/23 at Weill Cornell. Patients on NAT who did not complete treatment were excluded. RCB score (pCR/0, 1, 2, 3) was calculated from surgical resection pathology reports using the MD Anderson RCB Calculator [15, 16]. Baseline laboratory data were obtained from clinical records. Associations between NLR and PLR with RCB levels were examined using the non-parametric Kruskal-Wallis test (if >2 levels), the Wilcoxon rank sum test (if 2 levels), or the Spearman’s rank correlation. NLR and PLR were also correlated with PFS (measured from pembro start date to date of progression) in metastatic patients. PFS data for metastatic patients without interval imaging since pembro initiation were censored at the date of data analysis, and PFS was not calculated for patients who received only 1 cycle of pembro. Results: We identified 63 eligible patients: 24 were excluded due to lack of TNBC diagnosis (n=20), or because they only received adjuvant pembro (n=4). Of the 39 remaining patients, 5 did not complete NAT and 2 had missing data, leaving 32 evaluable patients (17 in the NAT cohort and 15 in the mTNBC cohort). Overall, the median age was 51 years, and 16% were Hispanic, 11% Asian, 57% non-Hispanic, 24% Black, and 65% Non-Black. Neoadjuvant cohort: The pCR rate was 30%. Patients with both NLR ≥ 5 and PLR ≥ 200, had a pCR rate of 0%; in patients with NLR < 5 or PLR < 200, pCR rate was 43%. Median NLR in pCR vs. non-pCR groups was 1.91 [IQR: 1.72- 2.73] vs. 2.44 [IQR: 1.97- 5.24] (p=0.3), with trends towards significance when NLR was compared between RCB levels >1 (2.67; IQR: 2.05, 6.49) vs. RCB levels ≤1 (1.91; IQR: 1.63, 2.58) (p=0.14). Mean PLR was higher in patients with RCB 2-3 (409) compared to those with pCR or RCB 1 (167) (p=0.146). Metastatic cohort: The overall mean PFS was 7.8 months. Visceral metastases were seen in 80% of patients (17% liver, 33% brain). In patients with brain mets, baseline NLR and PLR were higher than in the overall cohort (NLR, 4.86 vs. 2.78; PLR, 429 vs. 186). Conclusions: Our study is the first to evaluate NLR and PLR as response biomarkers in TNBC patients receiving pembro and is the first report of our real-world data of TNBC patients on pembro. In our neoadjuvant cohort, higher baseline NLR and PLR correlated with higher RCB levels, with a trend towards statistical significance. Further investigation and correlation of NLR and PLR with tumor microenvironment features will help to clarify these relationships. Citation Format: Ashley Schreier, Roberta Zappasodi, Inna Serganova, Laura Munoz Arcos, Xi Kathy Zhou, Massimo Cristofanilli, Eleni Andreopolu. Predictive value of neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios in patients with triple negative breast cancer treated with pembrolizumab [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 PO5-25-04.

Abstract PO3-16-05: Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development

Abstract Early detection of triple-negative breast cancer (TNBC) patients and their recurrence prediction are daunting tasks. We aim to develop mitochondrial DNA (mtDNA) based biomarkers to improve the management strategies of TNBC patients. We have undertaken mitochondrial genome (MG) enrichment and next-generation sequencing approaches to map the entire MG in 73 samples (64 tissues, 9 extracellular vesicle samples) from 32 highly-aggressive TNBC patients. Measurement of mtDNA and cardiolipin contents, in concert with NDUFB8 and SDHB proteins’ expression, was carried out in tumors and matched extracellular vesicles (EVs) of all the above TNBC patients. Overall, we identified 168 nonsynonymous mtDNA mutations, of which 73% (123/186) were coding and 27% (45/168) were non-coding. Twenty percent of mutations were nucleotide transversions. Respiratory complex I (RCI) appears to be the key target, which harbored 44% (74/168) of the overall mtDNA mutations. A panel of 11 hotspot mtDNA mutations was identified among 19-38% TNBCs, each of which was detectable in the matched EV samples with 82% specificity. Overall, 38% of the metastatic tumor-signature mtDNA mutations were detectable in matched EVs of the TNBC patients. An appreciable number of mtDNA mutations were homoplasmic (18%, 31/168), novel (14%, 23/168), and potentially pathogenic (9%, 15/168). The overall and RCI-specific mtDNA mutational load was recorded to be higher in African American (AA) compared to European American (EA) women with an exclusive abundance of respiratory complex (RC) protein NDUFB8 (RCI) and SDHB (RCII) therein. Increased mtDNA (p < 0.0001) content was measured in both tumors and matched EV samples along with an abundance of cardiolipin (p=0.0001) content in the EVs of the TNBC patients. Lethal tumor-signature mtDNA mutation detection and measurement of altered mtDNA and cardiolipin contents in the EVs at an early time point bear the potential to formulate noninvasive early detection and recurrence prediction strategies in TNBCs to improve the overall survival of the patients. Citation Format: Kunwar Vikramdeo, Shashi Anand, Sarabjeet Sudan, Paramahansa Pramanik, Seema Singh, Andrew Godwin, Ajay Singh, Santanu Dasgupta. Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development [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 PO3-16-05.

Abstract PO1-28-09: Rethinking the value of pathologic complete response rate for the survival outcomes of early triple-negative breast cancer treated with neoadjuvant chemotherapy: a systematic review and network meta-analysis

Abstract Background: Neoadjuvant chemotherapy (NACT) has been more frequently used in breast cancer, but it’s controversial whether NACT or adjuvant chemotherapy (ACT) is more effective in improving survival outcomes for triple-negative breast cancer (TNBC) patients. Recent studies also proposed pathologic complete response (pCR) could not be a robust surrogate for survival outcomes in certain types of breast cancer. To address these questions, a general meta-analysis and a network meta-analysis (NMA) were conducted to compare the survival outcomes of patients with pCR, non-pCR after NACT and ACT. The cut-off pCR rate was also declared to indicate when NACT produces equivalent survival outcomes to ACT (Registration: PROSPERO CRD42022336732). Methods: Databases including PubMed, Embase, Web of science and Cochrane library were searched up to June 2023 to investigate studies comparing NACT and ACT, as well as randomized controlled trials (RCTs) comparing different regimens in NACT or ACT settings in early operable TNBC patients. Heterogeneity was assessed using χ² based Q-test and I², combined with hazard ratios (HRs) with 95% confidence intervals (CI) computed for overall survival (OS), and disease-free survival (DFS, or event-free survival). The NMA with a Bayesian framework was conducted using both random and fixed effect model. The Weighted Least Square method and the Least Absolutely Deviation method were used to determine the cut-off pCR rates of OS and DFS. Results: A total of 35 studies involving 21 RCTs and 34143 TNBC patients were included. The general meta-analysis comprised 14 cohort studies. Eight high-quality cohort studies with propensity score matching and 21 RCTs were included in NMA. The pooled pCR rate for NACT was 42.2% (95% CI 37.8%-46.8%). Overall, the NACT cohort showed significantly worse OS and DFS than ACT cohort (HR=1.67, 95% CI 1.22-2.31; HR=1.37, 95% CI 1.14-1.64). However, patients with pCR after NACT showed improved OS than ACT (HR=0.58, 95% CI 0.50-0.66), while the patients without pCR after NACT had pooper OS than ACT (HR=2.03, 95% CI 1.88-2.19). As for DFS, there was no statistically difference between pCR after NACT and ACT groups (HR=1.04, 95% CI 0.63-1.73), while patients without pCR after NACT had significantly worse DFS than ACT (HR=2.65, 95% CI 1.98-3.55). From the NMA, the OS and DFS were better when combining modern target therapy such as Bevacizumab, PARPi, and PD-1/PD-L1 inhibitor (Figure 1), but not achieving pCR completely negated the benefits of newer drugs. Adding adjuvant capecitabine to patients not achieving pCR seemed to favor the OS. It was predicted that when the pCR rates were 64.7% and 46.9% (Figure 2), respectively, the OS and DFS of patients treated with NACT would be the same as those of patients treated with ACT. Conclusions: Higher pCR rate after NACT was associated with improved OS compared with ACT in TNBC patients. However, failure to achieve pCR after NACT resulted in worse survival outcomes than ACT. NACT had similar survival outcomes with ACT only when the pCR rate was at least 46.9%. The development of effective NACT regimens is beneficial for breast cancer patients. Figure 1. Forest plot of the Hazard Ratio for OS and DFS in TNBC patients pCR: pathological complete response; Bev: Bevacizumab; Pt: platinum; NACT: neoadjuvant chemotherapy; ACT: adjuvant chemotherapy; vel: veliparib; PDi:PD1/PDL1 inhibitor; X: capecitabine Figure 2. The cut-off value of pCR when NACT and ACT have the same Hazard Ratio based on OS and DFS. pCR: pathological complete response; Bev: Bevacizumab; Pt: platinum; NACT: neoadjuvant chemotherapy; ACT: adjuvant chemotherapy Citation Format: Xiaoying Qiao, Shu Wang, Taobo Hu, Baosheng Liang. Rethinking the value of pathologic complete response rate for the survival outcomes of early triple-negative breast cancer treated with neoadjuvant chemotherapy: a systematic review and network meta-analysis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-28-09.

Abstract PO5-03-05: Rethinking the value of pathologic complete response rate for the survival outcomes of early triple-negative breast cancer treated with neoadjuvant chemotherapy: a systematic review and network meta-analysis

Abstract Background: Neoadjuvant chemotherapy (NACT) has been more frequently used in breast cancer, but it’s controversial whether NACT or adjuvant chemotherapy (ACT) is more effective in improving survival outcomes for triple-negative breast cancer (TNBC) patients. Recent studies also proposed pathologic complete response (pCR) could not be a robust surrogate for survival outcomes in certain types of breast cancer. To address these questions, a general meta-analysis and a network meta-analysis (NMA) were conducted to compare the survival outcomes of patients with pCR, non-pCR after NACT and ACT. The cut-off pCR rate was also declared to indicate when NACT produces equivalent survival outcomes to ACT (Registration: PROSPERO CRD42022336732). Methods: Databases including PubMed, Embase, Web of science and Cochrane library were searched up to June 2023 to investigate studies comparing NACT and ACT, as well as randomized controlled trials (RCTs) comparing different regimens in NACT or ACT settingsin early operable TNBC patients. Heterogeneity was assessed using χ² based Q-test and I², combined with hazard ratios (HRs) with 95% confidence intervals (CI) computed for overall survival (OS), and disease-free survival (DFS, or event-free survival). The NMA with a Bayesian framework was conducted using both random and fixed effect model. The Weighted Least Square method and the Least Absolutely Deviation method were used to determine the cut-off pCR rates of OS and DFS. Results: A total of 35 studies involving 21 RCTs and 34143 TNBC patients were included. The general meta-analysis comprised 14 cohort studies. Eight high-quality cohort studies with propensity score matching and 21 RCTs were included in NMA. The pooled pCR rate for NACT was 42.2% (95% CI 37.8%-46.8%). Overall, the NACT cohort showed significantly worse OS and DFS than ACT cohort (HR=1.67, 95% CI 1.22-2.31; HR=1.37, 95% CI 1.14-1.64). However, patients with pCR after NACT showed improved OS than ACT (HR=0.58, 95% CI 0.50-0.66), while the patients without pCR after NACT had pooper OS than ACT (HR=2.03, 95% CI 1.88-2.19). As for DFS, there was no statistically difference between pCR after NACT and ACT groups (HR=1.04, 95% CI 0.63-1.73), while patients without pCR after NACT had significantly worse DFS than ACT (HR=2.65, 95% CI 1.98-3.55). From the NMA, the OS and DFS were better when combining modern target therapy such as Bevacizumab, PARPi, and PD-1/PD-L1 inhibitor, but not achieving pCR completely negated the benefits of newer drugs. Adding adjuvant capecitabine to patients not achieving pCR seemed to favor the OS. It was predicted that when the pCR rates were 64.7% and 44.6%, respectively, the OS and DFS of patients treated with NACT would be the same as those of patients treated with ACT. Conclusions: Higher pCR rate after NACT was associated with improved OS compared with ACT in TNBC patients. However, failure to achieve pCR after NACT resulted in worse survival outcomes than ACT. NACT had similar survival outcomes with ACT only when the pCR rate was at least 44.6%. The development of effective NACT regimens is beneficial for breast cancer patients. Citation Format: Xiaoying Qiao, Taobo Hu, Baosheng Liang, Shu Wang. Rethinking the value of pathologic complete response rate for the survival outcomes of early triple-negative breast cancer treated with neoadjuvant chemotherapy: a systematic review and network meta-analysis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-03-05.

Abstract PO2-13-07: SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT

Abstract Objectives/Rationale: Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for disease recurrence and death. While multiple prognostic biomarkers associated with response to NAST have been identified, adaptive resistance mechanisms to NAST remain poorly understood. Identifying and characterizing adaptive resistance mechanisms can illuminate novel therapeutic approaches and improve outcomes for TNBC patients with RD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with RD) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. Comparative marker selection analysis was performed by computing the two-sided paired samples t-test for each gene followed by pre-ranked gene set enrichment analysis (GSEA) amongst 23,797 annotated gene sets. Gene sets with false-discovery rate (FDR) corrected P values < 0.001 were determined to be significant. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients with RD (N=27 from NeoSTOP and N=31 from NeoPACT). 165/23,797 gene sets were significantly enriched in post-treatment compared to pre-treatment samples. SHEN_SMARCA2_TARGETS_UP (M29) was among the top five significantly enriched gene set and was selected for further analysis based on the known role of the SWI/SNF complex in cell survival and the availability of clinically viable SMARCA2 degraders. Pre-treatment SMARCA4 expression was associated with pCR, with lower median expression noted in patients with RD compared to patients who achieved pCR (P=0.04). Pre-treatment SMARCA2 expression was not associated with pCR (P=0.48). On paired sample analysis there was pronounced downregulation of SMARCA4 in post-treatment compared to pre-treatment samples in all patients (mean Z=-1.46, P< 0.0001), in patients treated with chemotherapy (mean Z=-1.65, P< 0.0001), and in patients treated with chemoimmunotherapy (mean Z=-1.28, P< 0.0001). Although there was no difference in SMARCA2 expression between paired pre- and post-treatment samples (mean Z=0.03, P=0.83), there was enrichment of SMARCA2 target expression on GSEA analysis (NES=2.63, FDR q< 0.00001) indicating that SMARCA2 activity is compensating for therapy-induced SMARCA4 loss. Conclusions: We identified 165 gene sets that were significantly enriched in paired post-treatment compared to pre-treatment TNBC samples. Altered activity of the SMARCA2 subunit of the SWI/SNF chromatin remodeling complex was identified as one of the top hits. SMARCA2 and SMARCA4 are mutually exclusive subunits of SWI/SNF, and inactivation of both SMARCA2 and SMARCA4 is synthetic lethal. We observed an association of lower pre-treatment SMARCA4 expression with resistance to NAST and pronounced therapy-induced downregulation of SMARCA4 and induction of SMARCA2 target genes in post-treatment compared to pre-treatment samples, suggesting that SMARCA2 compensates for adaptive SMARCA4 loss. SMARCA2 degraders are in early-phase clinical trials and are known to induce synthetic lethality in SMARCA4-deficient cells. Our results identify adaptive loss of SMARCA4 and synthetic lethal targeting of compensatory SMARCA2 as an attractive therapeutic target in TNBC patients with RD after neoadjuvant chemotherapy or chemoimmunotherapy. Citation Format: Shane Stecklein, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT [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 PO2-13-07.

Abstract PO2-03-05: Neoadjuvant anthracycline followed by Toripalimab combined with nab-paclitaxel in patients with stage IIA-IIIC triple negative breast cancer (NeoTENNIS): efficacy and safety results of a phase II study

Abstract Background: Immune checkpoint inhibitors(ICIs)have shown promising antitumor activity in triple negative breast cancer (TNBC) patients. Toripalimab, a novel PD-1 antibody, has been approved for the treatment in multiple solid tumors. However, the neoadjuvant use of toripalimab with chemotherapy has not been evaluated in TNBC patients. In addition, whether chemotherapy could sensitize the ICI treatment has not drawn conclusion yet. In this phase II trial, a sequential use of chemotherapy and anti-PD1 therapy was given in a neoadjuvant setting. The efficacy and safety of toripalimab were evaluated. Patients and methods: Female patients with histologically confirmed stage IIA to IIIC TNBC were included. Eligible patients received neoadjuvant therapy with four cycles of epirubicin-cyclophosphamide every 2 weeks followed by toripalimab (240 mg) every 3 weeks plus nab-paclitaxel weekly for 12 weeks. The primary endpoint was pathologic complete response (tpCR; ypT0/is ypN0) rate. Key secondary endpoints were breast pCR (bpCR; ypT0/is) rate, biomarker analysis, and safety. Results: Among 70 enrolled patients (median age, 51 years; 62.9% stage III), 66 patients completed study treatment without progression and received surgery after study treatment. Overall, the percentages of patients with a tpCR and bpCR were 55.7% (39 of 70 patients) and 58.6 % (41 of 70 patients), respectively. For women with CD8–positive and negative tumors, the tpCR rates were 66.0% and 30.0% (P = 0.006), respectively. Significant upregulation of CD8 positive cells after 4 cycles of epirubicin-cyclophosphamide induction chemotherapy (P = 0.011) were shown in sequential biopsy samples. Grade 3 or 4 treatment-related adverse events (TRAEs) occurred in 30 patients (42.9%), the most common of which were neutropenia (15.7%), leukopenia (14.3%) and alanine aminotransferase increase (4.3%). Most common immune-related AEs were hepatobiliary disorders (Grade 3, 7.2%), creatinine increase (all Grade 1-2, 2.8%) and hypothyroidism (all Grade 1-2, 2.8%). Conclusions: The addition of toripalimab to neoadjuvant chemotherapy is efficacious and safe in patients with locally advanced TNBC. Anthracycline-based chemotherapy could sensitize the ICI treatment with upregulated tumor immune response. The event-free survival results are expected with further follow up. Mechanisms of chemotherapy sensitization are still under investigation (ClinicalTrials.gov number: NCT04418154). Citation Format: Min He, Linxiaoxi Ma, Shuang Hao, Benlong Yang, Bingqiu Xiu, Ya-Yun Chi, Ruo-Hong Shui, Zhong-Hua Wang, Zhi-Ming Shao, Jiong Wu. Neoadjuvant anthracycline followed by Toripalimab combined with nab-paclitaxel in patients with stage IIA-IIIC triple negative breast cancer (NeoTENNIS): efficacy and safety results of a phase II 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 PO2-03-05.

Abstract PO3-13-06: TILs are an independent prognostic factor in early-stage TNBC patients achieving pathologic complete response after neoadjuvant chemotherapy

Abstract Introduction: In patients with triple negative breast cancer (TNBC) the achievement of pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) is associated with better long-term clinical outcomes. However, the attainment of pCR does not guarantee cure, since ~ 10% of patients undergo disease relapse. Higher levels of tumor infiltrating lymphocytes (TILs) are associated with better prognosis in TNBC patients undergoing upfront surgery and in patients with residual disease after NACT. However, the prognostic role of TILs among TNBC patients undergoing pCR during NACT has not been investigated so far. Methods: We included 134 TNBC patients (ER cutoff< 10%) who achieved pCR after NACT. These cases were identified from a cohort of 348 TNBC patients who received NACT at Istituto Oncologico Veneto (Padova) and Istituto Nazionale Tumori (Milano) from April 2004 to April 2021. TILs were quantified in pre-NACT tumor biopsy according to International guidelines. We assessed the association between TILs and relapse-free survival (RFS) or overall survival (OS). Results: Main patients characteristics were: median age 48 years; ductal histotype 92.8%; histologic grade 3 95.2%; stage I 9.7%, stage II 71.6%, stage III 18.7%. Almost all patients received anthracyclines (98.5%) and taxanes (99.3%) as part of NACT for a median number of 4 cycles each; 55.2% of patients received carboplatin. At a median FU of 4.6 years, the 5-yr RFS and OS rates were 90.4% and 94.4%, respectively. As a continuous variable (1% increment), TILs were independently associated with better RFS and OS after adjusting for patient age and tumor stage: HR 0.90 (95%CI 0.83-0.98), p=0.015 and HR 0.89 (95%CI 0.80-1.00), p=0.041, respectively. The best prognostic TILs cut-off in this cohort was 20%. Patients with TILs>20% showed a 100% 5-yr RFS rate (0/63 events) vs 82.6% (12/71 events) for patients with TILs< 20% (log-rank p< 0.001). OS rates at 5 years were 100% and 90.1% for high vs low TILs (log-rank p=0.007). Conclusions: This is the first demonstration of an independent prognostic role of baseline TILs in TNBC patients achieving pCR after NACT. Baseline TILs may discriminate patients who are cured after achieving pCR from patients at risk of disease relapse despite the achievement of pCR. These latter may be candidate to treatment escalation in the context of clinical trials. Our work provides relevant data in the ongoing debate on the role of adjuvant therapy (primarily pembrolizumab) following pCR. Citation Format: Maria Vittoria Dieci, Claudio Vernieri, Davide Massa, Lorenzo Nicolè, Gaia Griguolo, Federica Miglietta, Andrea Vingiani, Riccardo Lobefaro, Fabio Girardi, Grazia Vernaci, Tommaso Giarratano, Carlo Alberto Giorgi, Giancarlo Pruneri, Matteo Fassan, Filippo De Braud, Valentina Guarneri. TILs are an independent prognostic factor in early-stage TNBC patients achieving pathologic complete response after neoadjuvant chemotherapy [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 PO3-13-06.

A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

Abstract PO1-03-08: Association of patient-reported triple-negative breast cancer (TNBC) knowledge with clinical trial participation

Abstract Background: Patients with TNBC are highly encouraged to participate in clinical trials given the lack of targeted treatment options for this breast cancer subtype. Yet little is known regarding barriers to trial participation in this population, including the impact of patients’ understanding about TNBC biology. We examined whether disease knowledge and other patient-level factors were associated with TNBC patients’ participation in trials. Methods: From a multicenter, prospective cohort study of patients enrolled from 2019-present with newly diagnosed TNBC (ER/PR ≤10% also eligible), we identified those with stage I-III tumors who completed at least one survey. Surveys assessed demographics, TNBC knowledge, risk perceptions, and participation in clinical trials. Patients were assigned to Cohort A if surgery was the first intervention and Cohort B if neoadjuvant therapy was given. Medical records were reviewed to verify the accuracy of tumor characteristics reported by patients. Data was summarized with descriptive statistics and logistic regression was used to identify factors associated with trial participation. Results: Of the 137 patients included, median age was 56 years (IQR 46-63), 104 (82%) were non-Hispanic White, 10 (8%) Black, 5 (4%) Hispanic. and 110 (86%) had at least some college education. Overall, 55 (41%) had stage I tumors, 65 (48%) stage II, and 14 (10%) stage III. Among 116 patients with available clinical trial participation data, 55 (47%) reported trial participation, 59 (51%) reported no trial participation, and 2 (2%) were unsure. The majority (36/55, 65%) of trial participants were in Cohort B. Among 129 patients with TNBC knowledge data, 82 (64%) and 73 (57%) correctly reported their tumor stage and grade, respectively. Most (104/129, 81%) knew TNBC was defined as ER/PR/HER2-negative, but only 16% (21/128) knew the ER/PR-negative cut off was ≤1%. Of 98 patients with risk perception data, 30 (31%) estimated their likelihood of recurrence within the next 5 years as 0%, 60 (61%) between 10-50%, and 8 (8%) between 60-100%. When asked if TNBC patients who remain cancer-free after 3 years will most likely not experience recurrence, 44 (45%) said “True,” 13 (13%) said “False,” and 40 (41%) were unsure. On univariate analysis, trial participation was less likely in Cohort A patients (OR 0.42, 95% CI 0.20 – 0.89, P=0.02) and more likely in stage II patients (vs. stage I, OR 2.43, 95% CI 1.09 – 5.39, P=0.03). On multivariable analysis, patients with at least some college education had higher odds of trial participation compared to patients with a college degree or higher (OR 3.21, 95% CI 1.11 – 9.28, P=0.03). No associations were found for other factors, including TNBC knowledge. Conclusions: About half of TNBC patients surveyed reported participating in clinical trials. Most of these patients were undergoing neoadjuvant therapy, likely reflective of greater study availability in this setting. While TNBC knowledge was not significantly associated with trial participation in this small sample, a considerable number of patients reported their tumor characteristics incorrectly and/or appeared to underestimate their recurrence risks. This highlights the need for enhanced informational support for TNBC patients during treatment decision-making. More research is needed to understand barriers to trial participation in this population to leverage patient engagement and accelerate progress towards more effective treatments for TNBC. Citation Format: Megan Tesch, Noah Graham, Sean Ryan, Melissa Hughes, Eileen Wrabel, Nadine Tung, Steve Lo, Thomas Fynan, Meredith Faggen, Natalie Sinclair, Maria Constantinou, Sarah Sinclair, Nabihah Tayob, Nancy Lin, Ann Partridge, Eric Winer, Sara Tolaney, Ana Garrido-Castro. Association of patient-reported triple-negative breast cancer (TNBC) knowledge with clinical trial participation [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 PO1-03-08.

Abstract PS16-09: Analysis of the Radiosensitivity Index/Genomic-Adjusted Radiation Dose (RSI-GARD) in Paired Pre- and Post-Treatment TNBC Samples: Implications for Biomarker-Guided Radiotherapy

Abstract Objectives: Adjuvant radiotherapy is an important component of curative treatment for triple-negative breast cancer (TNBC). While there are several accepted variations in nominal dose and fractionation, these regimens are generally felt to be radiobiologically equivalent. Molecular radiosensitivity biomarkers have the potential to allow tailoring of physical radiotherapy dose for individual patients. The Radiosensitivity Index (RSI)/Genomic-Adjusted Radiation Dose (GARD) is a gene expression signature that predicts intrinsic radiosensitivity across multiple malignancies, with initial studies demonstrating clinical utility of RSI-GARD in TNBC. Since most TNBC patients will receive neoadjuvant systemic therapy, we sought to understand how neoadjuvant therapy-induced molecular adaptations could alter intrinsic radiosensitivity and RSI-GARD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with residual disease (RD)) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. The RSI was calculated according to the published algorithm using normalized expression of the 10 genes in this signature (AR, JUN1, STAT1, PRKCB, RELA, ABL1, SUMO1, PAK2, HDAC1, and IRF1). The resulting RSI score was substituted as the surviving fraction (SF) using the linear quadratic model of radiation-induced cellular lethality assuming a fractional dose of 2.0 Gy and a constant β=0.05 Gy−2 to derive a patient-specific α, which is a coefficient modifying dose-dependent cellular lethality resulting from binary mis-repair of double-strand breaks (DSBs) arising from a single particle track. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients (N=27 from NeoSTOP and N=31 from NeoPACT). One NeoPACT patient had a negative RSI, which led to an undefined α. Under the assumptions of the RSI-substituted LQ model, the median α/β ratio (reflecting sensitivity to fractionation) for all pre-treatment samples was 11.7 Gy. TNBC patients who achieved pCR had a significantly higher pre-treatment α (i.e., higher intrinsic radiosensitivity) compared to patients who had residual disease (P=0.04), and this trend was the same for patients treated with chemotherapy (P=0.31) or chemoimmunotherapy (P=0.05). Importantly, α did not change significantly between paired pre- and post-treatment samples (median change post- vs. pre-treatment=0.03 Gy−1 (range -0.25 to +0.24 Gy−1; P=0.26) in patients with RD, with no difference in subgroups treated with chemotherapy (P=0.99) or chemoimmunotherapy (P=0.10). Conclusions: We found that predicted intrinsic radiosensitivity using the RSI-GARD is associated with pathologic response to neoadjuvant systemic therapy, suggesting that this signature also predicts intrinsic chemosensitivity in TNBC patients. The modeled α/β ratio for all pre-treatment samples was 11.7 Gy, which is substantially higher than empirically determined α/β ratios in studies that were enriched for HR+/HER2- breast cancers. Globally, the RSI-GARD score was not impacted by neoadjuvant systemic therapy, with no significant change in the score in paired pre- and post-treatment samples. These results can inform future testing and implementation of RSI-GARD into prospective trials. Citation Format: Shane Stecklein, Julia White, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. Analysis of the Radiosensitivity Index/Genomic-Adjusted Radiation Dose (RSI-GARD) in Paired Pre- and Post-Treatment TNBC Samples: Implications for Biomarker-Guided Radiotherapy [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 PS16-09.

Abstract PO1-13-11: ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) poses significant clinical challenges due to the lack of targeted therapies and poor prognosis. Homologous recombination (HR) deficiency is a defining characteristic of TNBC and renders tumors susceptible to Poly (ADP-ribose) polymerase (PARP) inhibitors. However, not all TNBC patients respond to PARP inhibitors, highlighting the need to identify novel biomarkers to predict therapeutic response and improve treatment strategies. Methods: In this study, we investigated the role of zinc finger protein 689 (ZNF689) in DNA damage response (DDR) and its impact on PARP inhibitor sensitivity in TNBC. Using a comprehensive set of molecular and cellular techniques, including immunoprecipitation, in vitro and in vivo ubiquitination assays, protein expression analysis, and functional assays using TNBC cell lines, patient-derived organoids (PDOs), patient-derived xenograft (PDX) models, and cell line xenograft models, we elucidated the mechanistic role of ZNF689 in HR and PARP inhibitor response. Results: Our findings revealed ZNF689 as a novel player in DDR. ZNF689 is activated by ATM-mediated phosphorylation and recruited to DNA damage sites in an ATM-dependent manner. At the DNA double-strand break sites, ZNF689 collaborates directly with NBS1 to facilitate K63-linked ubiquitination of NBS1 via the E3 ligase SKP2. Furthermore, ZNF689-mediated NBS1 ubiquitination stabilizes the MRE11-RAD50-NBS1 (MRN) complex and feedbacks activation of ATM, therefore accelerating HR repair. Loss of ZNF689 results in HR deficiency, leading to increased sensitivity of TNBC cells to PARP inhibitors in both in vitro and in vivo models. Additionally, the combination of PARP inhibitors and paclitaxel demonstrated potential for improved efficacy in treating ZNF689-deficient TNBC. In our clinical analysis, we observed a positive correlation between ZNF689 expression and the levels of NBS1 and phosphorylated ATM in TNBC patient samples. Importantly, low ZNF689 expression was associated with a favorable response to PARP inhibitors in TNBC patients. Conclusions: Our study highlights the significant role of ZNF689 in HR and its impact on the response to PARP inhibitors in TNBC. These findings suggest ZNF689 as a promising therapeutic target for enhancing the effectiveness of PARP inhibitors in TNBC patients. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative 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 PO1-13-11.

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.

Abstract PO2-06-10: Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative breast cancer patients

Abstract Introduction: Immune checkpoint inhibitors (ICI) are an important therapeutic option for patients with triple negative breast cancer (TNBC). However, identification of patients most likely to respond is challenging. PD-L1 positivity by immunohistochemistry is the standard biomarker used for ICI therapy selection in TNBC. However, other biomarkers, such as analysis of the tumor microenvironment (TME) may be more accurate in predicting response. The XernaTM TME Panel uses RNA sequencing data and machine learning to analyze the TME, utilizing the angiogenic and immunogenic biology of the TME to classify tumors into four TME subtypes. In this study, the distribution of Xerna TME subtypes and associated genomic alterations in TNBC were investigated for their potential use in therapy selection. Methods: A total of 203 TNBC patient samples underwent tumor-normal whole-exome, whole-transcriptome sequencing testing with the OncoExTraTM assay. The whole-transcriptome expression data were analyzed using the Xerna TME Panel to assign each sample to one of four subtypes: Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A). The IA and IS subtypes both have high immune scores that may be particularly sensitive to ICI therapy. Actionable alterations, defined as those with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies, were also identified and associations across Xerna subtypes were explored. Results: Approximately half (100 of 203; 49.3%) of the patient samples had high (IA+IS) immune subtypes (Table 1). Targetable alterations associated with an FDA-approved therapy were present in 114 (56.2%) patients. No biomarkers were significantly associated (p < 0.05) with high (IA+IS) versus low (ID+A) immune scores. Biomarkers associated with ICI response, namely mismatch repair gene alterations (MSH2/3/6, MLH1/3, PMS1/2), high tumor mutational burden (TMB-high) and microsatellite instability were detected in only 6 (3.0%), 3 (1.5%) and 1 (0.5%) patient samples respectively, and all but 1, an MSH6 alteration, were in high immune subtype samples. Conclusions: The Xerna TME Panel classified 49.3% of TNBC patient tumors to IA or IS, suggesting they may respond to ICI therapy. Many (56.2%) patient tumors harbored alterations associated with FDA-approved therapies, providing the potential for novel combination therapies. These findings warrant further study and clinical validation in TNBC patients treated with ICI therapy. Table 1. Frequency of actionable biomarkers that were present in at least 10 (5%) TNBC patient samples. Citation Format: Gargi Basu, Janine Lobello, Snehal Thakkar, Jessica Aldrich, Matthew Halbert, Patrick Eimerman, Cynthia Flannery, Nishitha Therala, David Hall, Daniel Pointing, Lea Vohar, Roman Luštrik, Luka Ausec, Mark Uhlik, Seema Iyer, Laura Benjamin, Frederick Baehner. Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative breast cancer patients [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 PO2-06-10.

Abstract PO3-25-11: Unprecedented responses to neoadjuvant sequential administration of Tocilizumab followed by cisplatin/docetaxel for locally advanced triple negative breast cancer patients

Abstract Background: Triple negative breast cancer (TNBC) remains a challenge for clinicians due to its aggressive behavior and scarcity of effective treatment. Interleukin 6 (IL-6) is well known to contribute to poor therapeutic gain, tumor relapse and aggressive tumor growth. Here we have explored targeting the IL-6 signaling pathway by Tocilizumab (Actemra) as a new potential therapeutic approach for the treatment of TNBC. Methods: The study population includes thirty (30) patients with (T2-T4, N0-3) stage (IIB-III) TNBC treated uniformly with Tocilizumab followed with cisplatin/docetaxel according to a traditional 3+3 design using a sentinel patient at each cohort. Eligible patients received Tocilizumab on day 1 followed by cisplatin/docetaxel on day 2 every 4 weeks for a total of six cycles. The primary end points were pathological complete response in breast and axilla (pCR) (ypT0/ypN0) and assessment for tolerability/toxicities profiles. Additionally, the status of the BRCA1 and BRCA2 genes was determined in all patients. Results: Eligible patients who were enrolled in this phase I/II trial had a median age of 42 years, with 63% premenopausal and 67% had nodal involvement at base line. The median tumor size was 45 mm (range 25-150 mm), stage III A/B 26% and stage IIIC 50%. Germline BRCA1/2 mutations represented 27%. The overall pCR and (RCB class 0+1) were 66% and 69%, respectively. Interestingly, pCR reached 81% in axilla, and 100% in patients with tumors bearing BRCA1/2 mutations. Interestingly, tocilizumab affected the blood level of several pro-carcinogenic cytokines. Conclusions: To our knowledge this novel therapeutic strategy is the first to report the inhibition of a cytokine signaling (a new class of biological therapy) in combination with chemotherapy for the intricate and hard-to-treat TNBC patients with unprecedented responses and acceptable tolerability/toxicities, which merits further investigation. Pending Patents: US Patent Application No. 18/122,839 European Patent Application No. 23 162 694.6 Citation Format: Taher Al-Tweigeri, Noura Alraouji, Asma Tulbah, Syed Akhtar, Meshari Alzahrani, Saleh Najjar, Dahish Ajarim, Kauser Suleman, Adher Al Sayed, Abdelilah Aboussekhra. Unprecedented responses to neoadjuvant sequential administration of Tocilizumab followed by cisplatin/docetaxel for locally advanced triple negative breast cancer patients [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 PO3-25-11.

Abstract PO5-13-07: Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics

Abstract Background Early-stage triple-negative breast cancer (TNBC) is usually treated with neoadjuvant chemotherapy (NADC), but prognosis remains poor compared with other BC subtypes. Unfortunately, less than 30% of BC patients achieve pathological complete response (pCR) to NADC and the overall survival of TNBC when cancer spreads is estimated at 10-13 months. Complete response is associated with improved progression-free and overall survival rates, but more accurate prognostic markers are needed. In this study, we assessed the prognostic value of blood circulating metabolites by using targeted-based metabolomics. Methods Patients received standard neoadjuvant chemotherapy with epirubicin plus cyclophosphamide followed by paclitaxel. Blood samples were obtained upon completion of chemotherapy and before surgery. Pathological response to treatment was recorded according to the Miller & Payne system, and categorized as partial (MP 1, 2, & 3) or complete response (MP 4 & 5). Blood samples were collected in EDTA tubes, centrifuged and plasma was stored at -80°C. After QC standard addition and methanol extraction of proteins, four fractions of the resulting extract were analysed: two by reverse-phase/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one by reverse phase/UPLC-MS/MS with negative ion mode ESI, and one by hydrophilic interaction liquid chromatography/UPLC-MS/MS) by Metabolon using a Thermo Scientific Q-Exactive mass spectrometer with a heated electrospray ionization source and an Orbitrap mass analyser. Using Metabolon's hardware and software, raw data were extracted, identified, QC processed and quantified using the area under the curve (AUC). Identified compounds were compared with library entries of purified standards or recurrent unknown entities. The prognostic capacity of each metabolite was assessed using BRB ArrayTools. Selected metabolites were validated using a targeted approach using compound standards as follows. Plasma samples were re-analysed by UPLC/MS using a Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific) with positive and negative ionization mode ESI under polarity switching. Identification of compounds were performed by comparison of retention times (against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra. These analyses were carried out by MS-Omics (Denmark). Results Twenty patients treated in the Medical Oncology Unit of the University Hospital of Jaén were recruited. Median age was 50 (31-76). Twelve patients obtained a pathological complete response. Median follow-up after surgery was 25 months. Seven patients eventually had distant metastases (2 in the lungs, 1 in the brain, 1 in the bones and 3 multi-site). Metabolon untargeted analysis identified and quantified 985 metabolites, 789 after xenobiotics exclusion. Survival analysis identified 16 metabolites related with distant relapse (p< 0.05). The ten metabolites with lower p-value were selected for targeted validation. Ms-Omics pipeline allowed absolute quantification of 5 metabolites, 3 of them showing high correlation (R >0.7) between both measurements (targeted and untargeted). Validation of the prognostic value of the candidate metabolites is ongoing in a larger TNBC cohort. Conclusions Metabolomics is a useful tool for the detection of simply assessable and cost-effective prognostic biomarkers in TNBC. Easily monitoring the presence of minimal residual disease is worth to be settled up in the clinical practice for the most aggressive molecular subtype of breast cancer. In this work we have defined a set of metabolites that could predict distant relapse events in TNBC patients treated with neoadjuvant chemotherapy. Further analyses are being carried out to validate the prognostic value of the proposed candidate metabolites. Citation Format: Pedro Sánchez-Rovira, Rocio Urbano-Cubero, Angelo Gamez Pozo, Carmen González-Olmedo, Alicia Cano-Jimenez, Andrea Zapater- Moros, Leticia Díaz-Beltrán, Lucia Trilla-Fuertes, Mariana Díaz-Almirón, Enrique Espinosa, Pilar Zamora, Juan Angel Fresno Vara. Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-07.

Abstract PO1-06-08: Treatment utilization by race and insurance type among triple negative breast cancer patients

Abstract Introduction Triple negative breast cancer (TNBC) is associated with poorer prognosis and its lower long-term survival rates disproportionately affect people of racial and ethnic minority groups. Ensuring timely and effective treatment for all patients regardless of race or insurance status is critical. The objective of this study is to describe treatment patterns by line of therapy, race, and insurance type to identify barriers to accessing healthcare using a physician survey and patient-chart review. Methods This study employed a 60-minute physician survey and linked retrospective chart review of patients’ charts conducted June-July 2022. Participating oncologists across the US (34% Central; 19% Southeast; 18% Northeast; 21% West; 9% East Coast) were required to be board-certified, in practice 3 to 30 years post-residency, and managing at least 7 patients with TNBC in the past 3 years. Oncologists extracted patient-level data from 2-4 patient records for the chart review. A convenience sample of charts oversampled patients on Medicaid, Black and Latina patients, and patients with later stage TNBC to allow for robust disparities analysis. Data collection included provider characteristics, treatment by line of therapy (LOT), rationale for treatment decisions, barriers experienced in accessing care, and support services used. Results Participating oncologists (n=101, 73% male; 62% community vs 39% academic setting) provided data on 283 patients with TNBC [99% female; median age = 46.0 years; race = Black (42%), Latina (31%), White (25%) or Other (2%); geographic location = Urban (53%), Suburban (39%), Rural (8%)]. White patients were more likely to be covered by private insurance (73%) compared to Black (25%) and Latina patients (32%). Black and Latina patients were more likely to be covered by Medicaid (White, 16%; Black, 71%; Latina, 52%). Over half of patients needed prior authorization for first-line (1L) treatment; mean wait time for prior authorization was 1.5 weeks. Black patients (58%) were more likely to require prior authorization in 1L as compared to White patients (49%); There were no significant differences in prior authorization requirements based on type of insurance (Medicaid, Medicare, Private/Commercial Insurance). Of the 283 patients treated with 1L therapy, 111 patients received second-line (2L) treatment [Black (n=52, 44%), Latina (n=29, 32%), White (n=26, 37%), Other (n=4, 71%)], and 45 patients received third-line (3L) treatment [Black (n=25, 21%), Latina (n=9, 10%), White (n=9, 13%), Other (n=2, 33%)]. Chemotherapy was the most common 1L (75%) and 2L (51%) treatment. Targeted therapy (TT), defined as immunotherapy, hormone therapy or antibody–drug conjugates, was most common in 3L (64%). A smaller proportion of Black patients who progressed to 3L received TT (n=13, 52%) compared to White (n=8, 89%) and Latina (n=7, 78%) patients. A larger proportion of patients with private/commercial insurance (n=18, 95%) received TT in 3L as compared to patients with Medicaid (n=10, 42%) or Medicare (0%). Conclusions Findings indicated disparities both in pre-treatment barriers (specifically prior authorization) and the utilization of targeted therapy in later lines of therapy among Black patients. Although treatment patterns did not seem to differ by race in early lines of therapy, numerical differences in treatment patterns by race and insurance type emerged in later lines of therapy. Given the poor prognosis for patients diagnosed with TNBC and its disproportionate impact on communities of color, future policy efforts should be focused on achieving equity in treatment initiation and use of TT in TNBC patients. Citation Format: Natalia Sadetsky, Langa Bakhuluma-Ncube, Emily Fox, Laura Okpala, Matt O'Hara, Jennifer Parr. Treatment utilization by race and insurance type among triple negative breast cancer patients [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 PO1-06-08.

Abstract PO5-03-08: Detection and quantification of triple-negative breast cancer (TNBC) across ethnicities through analysis of cell-free DNA (cfDNA) methylation

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype contributing to race-related outcome disparities in breast cancer. These disparities are partly explained by the higher burden of TNBC in Black women, less access to new mammography technologies, and further compounded by limited efficacy of screening mammography in early detection of TNBC (Newman L & Mitchell E, J Natl Med Assoc 2023). To better understand these disparities, we compared results from the Galleri® multi-cancer early detection (MCED) test and the tumor methylated fraction (TMeF) tumor quantification algorithm in a population of TNBC patients across different stages and self-reported ethnicities (SRE). Methods: Within the case-control Circulating Cell-free Genome Atlas third substudy, we performed MCED testing and TMeF estimation in 507 women with breast cancer, 84 of whom had TNBC. The MCED test detects methylation patterns associated with circulating tumor DNA (ctDNA) to identify a common cancer signal and predict a cancer signal of origin. Tumor methylated fraction (TMeF) is an estimate of ctDNA abundance using machine learning models trained on whole-genome bisulfite sequencing of tumor biopsy tissue. We used SRE to compare results between non-Hispanic Black and White participants and those of other (Hispanic, Asian, Pacific Islander) or unreported ethnicities for both cancer detection by MCED test and ctDNA abundance estimation by TMeF. Results: In Black women, 19 (49%) of 39 diagnosed breast cancers were TNBC, compared to 58 (15%) of 396 breast cancers in White women and 7 (13%) of 52 breast cancers in women of other or unreported ethnicities. The MCED test did not detect Stage I TNBC (Table 1). In patients with Stage II-IV TNBC, the MCED test successfully detected a cancer signal in 16 (94%) of 17 Black women, 31 (72%) of 43 White women, and 6 (100%) of 6 women of other or unreported ethnicities (Table 1). When we examined the TMeF in these patients using a two-way ANOVA, we found a significant difference in mean TMeF by cancer stage (p < 0.001) but no significant difference in mean TMeF between SRE groups (p = 0.09) with the distribution of TMeF (Table 2). The ANOVA additionally found a significant interaction between cancer stage and SRE (p < 0.001) that could represent an impact of stage on TMeF that varied by SRE or differences in cancer stage distribution by SRE. Conclusions: In this pilot study, we observed that MCED testing was effective in detecting Stage II, III, and IV TNBC across SRE. These findings, in combination with the association of estimated ctDNA abundance with cancer stage but not patient SRE, provide hypothesis-generating evidence that analysis of methylation signals from across the genome can provide meaningful TNBC screening/detection information, independent of self-reported race/ethnicity. Table 1. MCED cancer detection rate by stage and SRE among TNBC patients. Table 2. Tumor methylated fraction (TMeF) by stage and SRE among TNBC patients. Citation Format: William Cance, Edison Liu, Margaret Antonio, Timothy Shaver, Stephannie Shih, Bong Chul Chu, Kathryn Kurtzman, Lisa Newman. Detection and quantification of triple-negative breast cancer (TNBC) across ethnicities through analysis of cell-free DNA (cfDNA) methylation [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 PO5-03-08.

Abstract PO5-13-05: Comparison of whole exome, whole transcriptome genomic profiling and targeted sequencing with 50-gene panels

Abstract Introduction Genomic profiling to identify targetable genomic alterations is the standard of care in the management of advanced cancers, including breast cancer (BC), and is recommended by ASCO provisional clinical guidelines. Genomic panels of ≤50 genes are generally less expensive than larger panels but may miss targetable alterations. This study compared a comprehensive tumor-normal whole-exome, whole-transcriptome panel to four 50-gene panels. In addition, for triple-negative BC (TNBC), the budget impact of using a comprehensive panel versus 50-gene panels was estimated. Methods Gene lists derived from three commercially available 50-gene panels (A, B and C) and a customized pan-cancer gene panel list (D) were used. The genes listed on the custom panel were chosen by medical experts. Samples from BC patients that underwent OncoExTraTM testing from May 2018 through March 2023 were included. The OncoExTra assay uses tumor-normal whole exome and whole transcriptome sequencing to detect somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. In this analysis, the focus was actionable biomarkers, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer. The frequencies of patients who would have been missed (no actionable alterations identified) using only the genes on each 50-gene panel were summarized. It was assumed that any alteration involving a listed gene would be detected, maximizing the potential utility of each panel. For TNBC, the budget impact of shifting the 20% of patients currently tested with 50-gene panels to OncoExTra testing was estimated using a previously published model. Results A total of 2161 BC patient samples were included. Across all samples, between 11.9% and 28.1% of patients would have been missed by restricting to only 50 genes (Table 1). The proportion of missed patients across panels was highest in TNBC and lowest in HER2+ cancers. Major drivers of missed patients included absence of FGF3/4/19 (A-D), MAP3K1 (A-D), BRCA1/2 (A, B, C), CCND1 (B, C, D), and PTEN (A, D). Across the four panels, patients were missed due to actionable alterations in 96 to 126 genes. Tumor mutational burden and microsatellite instability, which are not reported in 50-gene panels, were high in 35 (1.6%) and 7 (0.3%) BC patient samples, respectively. Within a 1-million-member plan, 44 TNBC patients would be eligible for testing. If all these patients were tested with OncoExtra rather than a 50-gene panel, the increase in per-member-per-month costs ranges from $0.0104-0.0172, driven predominantly by increased matched therapy costs. Between two and five TNBC patients would be eligible to receive a different treatment with a comprehensive panel. Conclusions In BC, between 11.9% and 28.1% of patients with actionable alterations would have been missed by restricting analysis to the 50 genes found in these panels. Using a comprehensive profiling assay such OncoExTra led to the identification of additional TNBC patients with actionable alterations at a minimal budget impact. Table 1. BC patients (pts) with actionable alterations missed by each 50-gene panel. NS = subtype not specified. Citation Format: Snehal Thakkar, David Hall, Jess Hoag, Cynthia Flannery, Nishitha Therala, Anson Tharayanil, Jesse Ortendahl, Gebra Cuyun Carter, Gargi Basu. Comparison of whole exome, whole transcriptome genomic profiling and targeted sequencing with 50-gene panels [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-05.

Abstract PO2-15-07: HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial

Abstract HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial Background: Biomarkers for de-escalation and escalation of systemic therapy in early-stage TNBC are needed. HER2DX is a standardized prognostic (risk-score) and predictive (pathological complete response [pCR]-score) assay based on clinical and gene expression-based data. Here we aimed to test the value of HER2DX assay in early TNBC. Methods: Standardized HER2DX was evaluated centrally using RNA from baseline FFPE tumor biopsies from the WSG-ADAPT-TN study (NCT01815242), a multicenter phase II trial that randomized 336 patients with stage I–III early TNBC to 12-weeks of nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 d1,8 every 3 weeks (arm A) versus nab-paclitaxel 125 mg/m2 plus carboplatin AUC2 day 1,8 every 3 weeks (arm B). The primary aim was to test the ability of HER2DX pCR and risk-score models to predict pCR (ypT0/isN0) and survival endpoints, respectively, such as invasive disease-free survival (iDFS), distant disease-free survival (DDFS) and overall survival (OS). Secondary objectives were to assess the association of the i) HER2DX immune signature score and ii) percentage (%) of stromal tumor infiltrating lymphocytes (sTILs) with efficacy endpoints. Uni- and multi-variable logistic regression and Cox models tested the association of each variable with each endpoint. Results: HER2DX was evaluated in 126 (37.5%) baseline pre-treatment tumors. Mean age was 52.1 (range 26-76). cT1 represented 40.5% of cases and 72.2% were cN0. Overall, pCR rate was 34.1% (95%CI 26.1 – 43.2) and median follow-up was 5.0 years. Median sTILs at baseline was 31.6% (range 0-90) and moderately correlated with the IGG signature (coefficient=0.57). The % of HER2DX high-, medium- and low-pCR groups was 41.3%, 47.6% and 11.1%, respectively. HER2DX pCR score (as a continuous variable) was significantly associated with pCR in univariate (odds ratio [OR per 10-unit increase]=1.31, 1.06-1.64, p=0.016) and after adjusting by treatment arm (OR=1.31, 1.06-1.65, p=0.015). Overall, the pCR rates in HER2DX pCR-high, -medium and -low groups were 46.2%, 26.7% and 21.4%, respectively (high vs medium/low OR=2.48, 1.17-5.34, p=0.018). In arm B (n=51), the pCR rates in HER2DX pCR-high, -medium and -low groups were 47.8%, 25.0% and 0.0% respectively (high vs medium/low OR=3.36, 1.02-12.0, p=0.051). In terms of the risk-score, the % of HER2DX low-risk and high-risk groups was 60.3% and 39.7%, respectively. HER2DX risk score as a continuous variable was significantly associated with iDFS, DDFS and OS (p< 0.001 in all univariate analysis), and after adjusting by variables such as arm and pCR status (all p< 0.05). sTILs were not associated with any survival endpoint (all p >0.35). The HER2DX immune signature was significantly associated with iDFS (p=0.012), DDFS (p=0.022) and OS (p=0.012). Conclusion: The HER2DX genomic test in TNBC provides valuable insights into the response and survival following neoadjuvant taxane-based chemotherapy in the absence of immunotherapy. The development of a tailored genomic assay for TNBC is currently in progress. Citation Format: Oleg Gluz, Fara Brasó-Maristany, Ulrike Nitz, Laia Paré, Matthias Christgen, Guillermo Villacampa, Benedetta Conte, Sherko Kuemmel, Ronald Kates, Cornelia Kolberg-Liedtke, Eva-Maria Grischke, Helmut Forstbauer, Michael Braun, Mathias Warm, John Hackmann, Christoph Uleer, Bahriye Aktas, Claudia Schumacher, Ana Vivancos, Rachel Wuerstlein, Joel S. Parker, Monika Graeser, Charles M. Perou, Christine zu Eulenburg, Patricia Villagrasa, Hans-Heinrich Kreipe, Aleix Prat, Nadia Harbeck. HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial [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 PO2-15-07.

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p< 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [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 PO3-25-07.