Early TNBC Research Articles

272P Modulation of systemic and intratumor metabolism predicts response to fasting-mimicking diet plus chemotherapy in patients with early TNBC

272P Modulation of systemic and intratumor metabolism predicts response to fasting-mimicking diet plus chemotherapy in patients with early TNBC

Circulating tumor DNA (ctDNA) to predict response to neoadjuvant chemotherapy (NAC) and prognosis in patients (pts) with early breast cancer (BC).

e12642 Background: pCR to NAC is an important predictor of clinical recurrence. Up to 16% of patients with pCR may experience disease recurrence, 45% of patients with RD do not. Longitudinal ctDNA testing offers a minimally invasive approach for monitoring treatment response EBC patients receiving NAC. We evaluated whether ctDNA status and dynamics can predict the tumor response to NAC and the risk of recurrence in patients with EBC. Methods: We conducted an observational, retrospective, single-center study to investigate whether ctDNA can predict tumor response to NAC and prognosis in BC patients. 74 pts received standard of care NAC;g 4 patients were treated in the adjuvant setting. Blood samples were collected at diagnosis from 44 pts (baseline) and longitudinally during treatment from 34 patients. Tumor response and progression outcomes were reported. A clinically validated, tumor-informed ctDNA assay (Signatera). ctDNA status and dynamics were correlated with pathologic and radiologic outcomes. Results: 29 TNBC pts were ctDNA+ at baseline, 6 were ctDNA- Of the 29 pts, early ctDNA clearance by week 6 of NAC was observed in 10 pts, weeks 7-16 in 9 pts, clearance post-surgery in 1, 3 pts remained ctDNA+ after surgery, serial ctDNA testing is underway for the remaining 6 pts. All TNBC patients who had radiological complete response (CR) had early clearance of ctDNA . All pts with persistent ctDNA positivity levels after completion of NAC had radiological partial response (PR). Of the 9 TNBC pts with undetectable baseline ctDNA, 33% achieved CR on MRI and 43% achieved pathologic CR. Of the 19 TNBC pts with ctDNA clearance in response to NAC, 89% (17/19) remained ctDNA- and with no evidence of disease (NED) (length of follow up). 1 pt initially cleared their ctDNA on NAC, turned ctDNA+ 4 weeks prior to radiological recurrence. 1 pt with ctDNA clearance on NAC remained ctDNA post-surgery but experienced recurrence. All (n=3) TNBC pts with persistent ctDNA-positivity levels after curative treatment (NAC and surgery) had disease recurrence. The remaining 6 pts ctDNA are ongoing NAC. Of the 6 pts with undetectable baseline ctDNA, 6/6 continued to be ctDNA- regardless of their disease status. At diagnosis, 8/9 ER+ ps were ctDNA+. 3/8 (38%) ER+ pts with positive baseline ctDNA had early clearance of ctDNA after NAC. All 3 had residual disease on pathology. 2/8 (25%) remained ctDNA+ after surgery and neither achieved radiologic CR or pCR. The remaining 3 pts are ongoing NAC. Conclusions: In early TNBC, ctDNA monitoring can be used to predict tumor response to NAC and tailor therapeutic regimens. ctDNA persistence can predict early recurrence and helps identify patients in which treatment escalation or enrollment on clinical trials is necessary. In ER positive breast cancer, and despite the small number of patients, ctDNA clearance does not predict response to NAC.

Treatment outcomes of the KEYNOTE-522 regimen in an ethnically diverse patient population: A real world experience.

e12663 Background: Pembrolizumab (pembro) in combination with neoadjuvant chemotherapy (NACT) became standard of care for high risk, early stage TNBC on July 26, 2021, as a result of the KEYNOTE-522 trial. Black women are disproportionately affected by TNBC with more advanced stage at diagnosis, however the KEYNOTE-522 did not collect data on race. This retrospective review evaluates response to pembro plus NACT in a racially diverse population to assess the generalizability of the trial findings to potentially under-represented patient populations. Methods: This is a single center, retrospective review of patients with stage II/III TNBC diagnosed between 08/2020 to 08/2023, treated with pembro plus NACT at Maimonides Medical Center, a safety-net hospital in Brooklyn, New York serving a diverse patient population. Exclusion criteria included age < 18 years, ER/PR/Her2 positivity, NACT without pembrolizumab and unreported race. Data were collected from electronic health records and analyzed using SPSS Statistics. Results: 50 patients met inclusion criteria, among whom 40 patients had complete information available for review. 52.5% (95% CI: 38% - 69%) of patients had a pCR. Nearly half of the patient population (47.5%) were African American. Subgroup analysis of ethnicity demonstrated a lower pCR amongst African American women, but this was not statistically significant (table). 5 patients had progression of disease with the development of distant metastasis (2 whilst on treatment and 3 following completion of therapy). 3 patients had grade 3/4 adverse events (pembro associated endocrine and cardiac dysfunction). Notably, lack of complete imaging response to treatment was significantly associated with lack of pCR (p=0.003). Conclusions: In this retrospective review of a racially diverse patient population, the pembrolizumab-NACT regimen was well tolerated with an acceptable side effect profile. While our rate of pCR is lower than the findings of the Keynote-522 trial; 52.5 % (95% CI: 38% - 69%) vs. 64.8% (95% CI: 59.9%-69.5%), our sample size is not large enough to detect a statistically significant difference. Sub-analysis of patient ethnicity demonstrated varying rates of pCR, however findings are limited by a small sample size. Additional data collection is in progress and future analysis will provide insight into a potential correlation between race and therapy response. [Table: see text]

Predictive markers of complete pathological response to neoadjuvant chemo-immunotherapy in triple negative breast cancer.

e12650 Background: Results from the Keynote 522 clinical trial, which played a pivotal role in evaluating the efficacy of neoadjuvant pembrolizumab in combination with chemotherapy (NAICT), showed a significant improvement in pathological complete response (pCR) rates compared with chemotherapy alone. To date, no significant markers have been identified to predict pathological complete response to NAICT. The primary objective was to identify markers, both molecular and clinical, predictive of pCR to NAICT in a real-world clinical setting. Methods: The retrospective analysis included 38 consecutively treated pts with early stage TNBC who received NAICT including pembrolizumab at the Institute of Oncology, Ljubljana (OIL) between 4 January 2022 and 17 October 2023. Data collected from electronic medical records included tumour size, nodal status, UICC stage, BRCA status, systemic immune-inflammation index (SII) before NAICT and before surgery, and frequency and grade of immune-related adverse events (iAEs) (according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5). In pts who completed surgical treatment, response to treatment was assessed by pathological examination of post-treatment surgical specimens. Descriptive statistical methods were used to summarise and describe the main characteristics. Pearson chi-squared test was used to analyse categorical variables and t-test to evaluate associations between continuous variables. Results: Of the 38 pts, 28 completed surgical treatment and were further analysed. Pathohistological examination of post-treatment surgical specimens revealed pCR in 11 pts (39.3%), with residual disease in 17 pts (60.7%). Of the 17 pts without pCR, 11 pts had RCB score 2 (64.7%) and 5 pts had score 3 (29.4%). The distribution of size, nodal status, UICC stage at diagnosis, BRCA status, frequency of iAEs in correlation with pCR is shown in the Table. A borderline correlation was found between pCR and SII at the start of pembrolizumab treatment (p=0.05). Age, tumour size, nodal status, UICC stage, BRCA status, AEs, SII before surgery were not found to be predictive of pCR. Conclusions: SII at the start of pembrolizumab was found to be a borderline significant marker predictive of pCR. Other characteristics evaluated did not correlate with pCR. [Table: see text]

Spatial transcriptomic and proteomic insight into Trop-2, HER2, and AR expression: A pathway to tailored therapies in triple-negative breast cancer.

e12576 Background: Triple-negative breast cancer’s (TNBC) dismal prognosis demands more effective therapy selection. Previously described molecular subtypes reveal its heterogeneity but lack spatial insight and are not currently used in clinical practice. Here, we aim to evaluate by immunohistochemistry (IHC) the expression of key biomarkers in TNBC and its correlation with spatial gene expression obtained by spatial transcriptomics (ST). Methods: We studied a retrospective cohort of 92 patients (pts) treated at the Institut Jules Bordet in Belgium with primary surgery for early TNBC. ST (Visium Spatial Gene Expression, 10X Genomics) was previously performed on fresh frozen surgical samples. Using serial sections of these samples, we performed duplex IHC staining for Trop-2/androgen receptor (AR) and HER2/Ki-67. IHC staining was scored using H-score (Trop-2), Allred score (AR), and current guidelines (Ki67/HER2), adding the ultra-low and null HER2 categories. Statistical analyses included descriptive statistics, Spearman correlation, Mann-Whitney test, and Kaplan-Meier with log-rank tests for estimating outcome distribution. Results: Of 92 pts included, 4 (4.3%), 25 (27.2%), 28 (30.4%), and 35 (38%) pts had HER2 2+, 1+, ultra-low, and null, respectively, tumors. Importantly, we observed a significant difference in tumor cell-derived ERBB2 gene expression of HER2 ultra-low versus HER2 null tumors ( p<0.0001), while there was no difference in ERBB2 expression between HER2 ultra-low and HER2 1+ tumors ( p=0.49). HER2 expression was not correlated with prognosis. Trop-2 expression by IHC was high, medium, and low in 67%, 23%, and 10% of pts, respectively. IHC expression correlated well with tumor-derived gene expression (r=0.54), with intrasample heterogeneity observed both in IHC and spatial gene expression. Trop-2 expression was prognostic, pts with high Trop-2 H-score tumors having significantly worse outcome ( p=0.021). In this cohort, 25%, 40.2%, and 34.8% of pts had AR Allred scores of 0-2, 3-5, and 6-8, respectively. Tumor AR gene expression was well correlated with IHC AR expression (67.7%). IHC AR expression was associated with the presence of adipose tissue in the stroma (r=0.23, p=0.025) but not with prognosis. Of note, we report for the first time, to our knowledge, for TNBC, AR expression both in tumor and stroma cells, both at the IHC and gene expression levels. Importantly, we could construct a classifier that identifies luminal androgen receptor tumors, using AR Allred score > 5 and Ki67 ≤ 40%, with a PPV of 93% and an NPV of 96%. Conclusions: ST data has the potential to improve easily accessible IHC classifiers, with the aim of refining patient selection for ADC and anti-AR therapies in TNBC.

A prospective phase II study of neoadjuvant therapy guided by personalized drug-testing on patient-derived tumor-like cell clusters for early breast cancer.

e12625 Background: Individual tumors of breast cancer patients possess unique characteristics that may influence their neoadjuvant therapy (NAT) response to specific cytotoxic agents and treatment choices. Therefore, it is essential to have a platform that can assist in the personalized selection of chemotherapy for individual patients. Previously, we reported an in vitro model called patient-derived tumor-like cell clusters (PTC), which can structurally and functionally recapitulate original tumors. Retrospective research has shown that PTC hold potential as reliable pre-clinical drug testing model for guiding NAT therapeutic options in early breast cancer patients. Methods: This prospective, phase II, open-label study aimed to validate the efficacy of drug screening on patient-derived tumor-like cell clusters (PTCs). TNBC and HER2 positive patients received neoadjuvant therapy based on subtype and personalized drug testing on PTCs. TNBC patients were treated based on drug testing, receiving either TA (taxanes combined with anthracyclines) or TP (taxanes combined with carboplatin). HER2 positive patients received treatment based on drug testing, including THP (taxanes, trastuzumab and pertuzumab) or TCbHP (taxanes, carboplatin, trastuzumab, and pertuzumab). Sample size calculation was performed using Fleming's two-stage design. For TNBC patients, if 10 or more out of 20 patients achieved pCR in the first stage, an additional 26 patients would be enrolled in the second stage. For HER2-positive patients, the sample size was same with TNBC patients. The study was approved by the Ethics Committee of Peking University People's Hospital in Beijing and was registered on ClinicalTrials.gov (NCT04750122, NCT04836156). This time, we report the results of the first stage. Results: Among 20 enrolled TNBC patients in the first stage, 15 achieved pCR with the rate of 75%. 17 patients (85%) had RCB 0-I score while 3 patients (15%) had an RCB-II score. 14 patients received TP as it demonstrated greater sensitivity in vitro. Out of these 14 patients, 10 patients (71.4%) achieved pCR. Six patients received TA as it was more effective in drug testing. Among these patients, 5 (83.3%) patients achieved pCR. Out of 20 enrolled HER2-positive patients, 16 achieved pCR, resulting in a pCR rate of 80%. Six patients received THP treatment based on their sensitivity to this regimen in drug testing. Among these patients, 5 (83.3%) achieved a pCR. 14 patients who were sensitive to TCbHP but not to THP in drug testing received TCbHP treatment. Among these patients, 11 (78.6%) achieved pCR. The non-pCR patients had 2 RCB-II score and the ORR was 100% for all 14 patients. Conclusions: Our results demonstrate that personalized drug testing on PTCs can effectively guide the choice of neoadjuvant therapy for early TNBC and HER2-positive breast cancer patients. Clinical trial information: NCT04750122 , NCT04836156 .

IMpassion132 double-blind randomised phase III trial of chemotherapy with or without atezolizumab for early relapsing unresectable locally advanced or metastatic triple-negative breast cancer

Immune checkpoint inhibitors improve the efficacy of first-line chemotherapy for patients with programmed death-ligand 1 (PD-L1)-positive unresectable locally advanced/metastatic triple-negative breast cancer (aTNBC), but randomised data in rapidly relapsing aTNBC are scarce. IMpassion132 (NCT03371017) enrolled patients with aTNBC relapsing <12 months after last chemotherapy dose (anthracycline and taxane required) or surgery for early TNBC. PD-L1 status was centrally assessed using SP142 before randomisation. Initially patients were enrolled irrespective of PD-L1 status. From August 2019, enrolment was restricted to PD-L1-positive (tumour immune cell ≥1%) aTNBC. Patients were randomised 1:1 to placebo or atezolizumab 1200 mg every 21 days with investigator-selected chemotherapy until disease progression or unacceptable toxicity. Stratification factors were chemotherapy regimen (carboplatin plus gemcitabine or capecitabine monotherapy), visceral (lung and/or liver) metastases and (initially) PD-L1 status. The primary endpoint was overall survival (OS), tested hierarchically in patients with PD-L1-positive tumours and then, if positive, in the modified intent-to-treat (mITT) population (all-comer patients randomised pre-August 2019). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR) and safety. Among 354 patients with rapidly relapsing PD-L1-positive aTNBC, 68% had a disease-free interval of <6 months and 73% received carboplatin/gemcitabine. The OS hazard ratio was 0.93 (95% confidence interval 0.73-1.20, P= 0.59; median 11.2 months with placebo versus 12.1 months with atezolizumab). mITT and subgroup results were consistent. Median PFS was 4 months across treatment arms and populations. ORRs were 28% with placebo versus 40% with atezolizumab. Adverse events (predominantly haematological) were similar between arms and as expected with atezolizumab plus carboplatin/gemcitabine or capecitabine following recent chemotherapy exposure. OS, which is dismal in patients with TNBC relapsing within <12 months, was not improved by adding atezolizumab to chemotherapy. A biology-based definition of intrinsic resistance to immunotherapy in aTNBC is urgently needed to develop novel therapies for these patients in next-generation clinical trials.

Abstract PS04-01: Clinical impact of timing of systemic therapy in patients with early triple negative breast cancer

Abstract Introduction TNBC is known for its aggressive nature, with early recurrence and limited treatment options beyond chemotherapy and recently immunotherapy. We aim to clarify, whether timing of systemic therapy has an impact on survival in patients with early TNBC. Methods Data from a large population-based regional cancer registry covering over 2.2 million people was used for evaluation, including women with diagnoses of TNBC between 2010 and 2018. Patients were categorized into subgroups according to the interval from TNBC pathologic diagnosis to the date of first administration of NACT (TTNC) or according to the interval from primary surgery to the date of first administration of adjuvant chemotherapy (TTAC). Univariable analyses of survival rates were conducted using the Kaplan-Meier method, the log-rank test was used for group comparisons. IBM SPSS Statistic 25 was used to perform statistical analysis, with hazard ratios, p-values, and 95% confidence intervals calculated for each model. All tests were two-sided, and significance was set at p &amp;lt; .05. Results We identified 37 382 patients with malignant neoplasms of the mammary gland. The final study cohort included 732 patients with early TNBC, of whom 43.6% were treated with NACT and 40.3% with adjuvant chemotherapy. Timing of NACT 270 patients with TNBC treated with NACT had a valid TTNC. Median follow up was 3.5 years. Patients with TTNC ≤14, ≤42, &amp;gt;42, and &amp;gt;56 days had estimated mean OS of 8.4, 6.9, 4.6, and 3.3 years, respectively. Patients with a TTNC ≤14 days were more likely to survive than patients with a TTNC ≥ 56 days (p= 0.054). Significance was barely not reached in the group of NACT. Timing of adjuvant chemotherapy 245 patients with TNBC treated with adjuvant chemotherapy had a valid TTAC. Treatment given within 22-28 days led to the best outcome with a mean OS of 10.2 years, while later treatment (29-35, 36-42 days, or &amp;gt;6 weeks) had significant reduced mean OS of 8.3, 7.8, and 6.9 years. Patients who received therapy within 22-28 days had significantly better survival than those who received therapy between 29-35 days (p=0.043) or after more than 43 days (p=0.033). Conclusion Although significance was barely not reached in the subgroup of patients treated with NACT, the results in this large population-based study indicate that there are critical time intervals for initiation of systemic therapy in patients with early TNBC, with reduced OS if NACT is applied later than 42 days after diagnosis or if adjuvant chemotherapy is applied later than 42 days after surgery. We recommend not to exceed the 6-week interval until the initiation of systemic therapy in patients with early TNBC. Citation Format: Maria Eleni Hatzipanagiotou, Miriam Pigerl, Michael Gerken, Sophie Räpple, Verena Zeltner, Madeleine Hetterich, Elisabeth C. Inwald, Monika Klinkhammer-Schalke, Olaf Ortmann, Stephan Seitz. Clinical impact of timing of systemic therapy in patients with early 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 PS04-01.

Abstract PO4-13-11: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results in early TNBC, many patients do not respond, and chemotherapy remains the main treatment. High recurrence rates in TNBC are in part due to its inherent molecular heterogeneity and sub-clonal diversity, whereby cells present in minority sub-populations escape therapeutic pressure. Recurrent genomic alterations are not selected for upon therapy resistance in neoadjuvant chemotherapy (NAC) resistant TNBC, however sub-clonal transcriptionally defined cells are “primed” to drive resistance and are pre-existing, suggesting phenotypic heterogeneity is governed by inheritable activation of epigenetically defined regulatory regions. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Driver DNA mutations and copy number alterations (CNA’s) were identified using a clinically validated targeted capture panel. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine meta-clusters across the dataset. A distinct immune-like MC was contributed by epithelial G1 cells, suggesting a cross-talk of epithelial cells with spatially proximal immune cells. Two MCs were dominated by cells from ChemoR patients and were defined by pathways related to hypoxia, EMT, and extra-cellular matrix signalling. Using scATAC profiles, we identified that the sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of distal enhancer chromatin modification in regulating MC transcription. None of the meta-clusters were dominated by tumours with specific genetic mutations or CNA’s. Spatial ATAC and RNA profiling of a subset of tumours identified spatial epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and associated with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommati, Shefali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance 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 PO4-13-11.

Abstract PO1-03-10: Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer

Abstract Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer Background: It has previously been shown that microbial metabolic pathways are differentially activated in immunotherapy resistance, hence can serve as potential targets for therapeutic intervention. With the advent of Immune Checkpoint Blockers in the neoadjuvant setting of locally advanced (LA) TNBC, we hypothesized that similar microbial metabolite mediated resistance may be at play. We are presenting the real world data for neoadjuvant treatment in early TNBC along with a subset of tumor metabolite analysis. Method: 35 patients who received neoadjuvant pembrolizumab and chemotherapy for LA TNBC were identified at Moffitt Cancer Center between July 2019 to January 2023. These included 22 Caucasian, 5 African American, 2 Hispanic and 6 patients with undisclosed race. The age range was 33-78 years. The weight and Body Mass Index range was 55.3 kg-127.3 kg and 20.96-45.43 kg/m2 respectively. Nine patients had stage III disease while 24 had stage II breast cancer. The response was assessed in 33 patients; 20 patients (60.6%) achieved pathological complete response (pCR) and 13 (39.4%) had residual disease (RD). 1 patient developed severe immune mediated toxicity with colitis, hepatitis, myasthenia gravis leading to death while 1 patient had disease progression leading to drug discontinuation. Six patients developed immune mediated adverse events. We conducted untargeted metabolomic profiling on 24 patients to identify differentially abundant metabolites associated with pCR (ypT0/is ypN0) vs RD; 16 vs 8 patients respectively. Formalin fixed paraffin embedded (FFPE) slides from tumor tissue vs tumor bed were retrieved and histopathological evaluation of tumor cells was performed. 20 μm sections of FFPE block were used for untargeted metabolomics via liquid chromatography/mass spectrometry. Among the 200 metabolites detected, 63 were differentially abundant between pCR and tumor samples with RD. After correcting for false discovery rate, we observed striking differential activation of two microbial metabolic pathways. Samples with RD had significantly elevated kynurenine (OR-3.64 X1034, P-1.94 X 10-74 and kynurenine/Tryptophan ratio). The tryptophan, kynurenine pathway has been known to be overexpressed in patients resistant to immunotherapy and modulated by the microbiome. Similarly, the purine metabolic pathway, specifically microbiota-derived immunostimulatory metabolites- inosine, hypoxanthine, uric acid were significantly depleted in patients with RD (Inosine OR-0.54, p-0.007, Hypoxanthine OR-0.5, p-0.00039, uric acid OR: 0.41, P-0.003). Previously Inosine has been shown to increase the anti-tumor effects of immune checkpoint blockade by enhancing CD8+ effector T-cell function. Hence depletion of inosine, hypoxanthine and uric acid signals lack of an effector CD8 T cell response. Conclusion: Despite being a small study, it corroborates pCR rates noted in KEYNOTE 522 in the real world setting but shows significant concern for serious immune mediated adverse effects. This highlights the importance of stratifying patients who might be resistant to ICB or develop significant toxicities, thus deriving little to no benefit from standard neoadjuvant treatment. This underscores the need to develop precision-based treatment algorithms. The study represents one of the first investigations of differential abundance of microbial metabolites with immunotherapy resistance in LA TNBC. Our results highlight two microbially mediated mechanisms that could be targeted to improve pCR rates in LA TNBC. We are working to identify microbial metabolic pathways associated with immune mediated toxicity. Citation Format: Humaira Sarfraz, Brian Czerniecki, Zena Jameel, Michael Jaglal, Mostafa Eysha, Turkan Ozkul, Jameel Muzaffar, Aditi Saha, Sara Sarfraz, Fatima Zahra, Min Liu, Khushali Jhaveri, Melissa Sur, Adrianna Bagos-Estevez, Shahla Bari. Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced 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 PO1-03-10.

Abstract PO5-03-07: Impact of age and stage on pathologic complete response rates in Black vs White patients with triple negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) accounts for approximately 10-15% of breast cancers. Women with TNBC have worse survival outcomes, increased rates of relapse, and distant metastasis as compared to women with non-TNBC. Regardless of subtype, Black women have worse breast cancer outcomes than White women. Early stage treatment of TNBC has focused on neoadjuvant therapy (NACT) with a goal of achieving a pathologic complete response (pCR), which is associated with longer event-free survival and overall survival in TNBC patients. NACT with pembrolizumab was presented at the San Antonio Breast Cancer Symposium in December 2019 and FDA approved in July 2021. The Keynote-522 Trial found that patients who received NACT + pembrolizumab were more likely to achieve pCR than women who received NACT + placebo, regardless of stage. The KEYNOTE-522 trial did not examine race or age differences in treatment efficacy. Since TNBC disproportionately affects younger women and Black women, optimizing pCR rates in these groups is essential. Our objective is to examine racial differences in pCR rates by cancer stage and age among patients with TNBC who completed NACT/pembrolizumab. Methods Patient records from Ochsner Health, a regional network, were reviewed for those with early stage TNBC who had completed treatment with NACT/pembrolizumab from January 2020 to January 2022. Exclusion criteria included no surgical treatment for TNBC, unknown receptor status, and age &amp;lt; 18 years-old. Chi-square and Fisher’s Exact tests were used to examine the relationship between race and pCR stratified by stage at diagnosis and age. Risk ratios and 95% confidence intervals were also estimated to examine the strength of these relationships. Results We identified 92 patients with TNBC who completed treatment with NACT/pembrolizumab and underwent surgery. 52 patients (56%) were Black and 40 patients (43%) were White. 53.85% of Black patients had pCR and 67.50% of White patients experienced pCR (p=0.19). Among patients with stage III disease, only 37.50% of Black patients experienced pCR compared to 69.23% of White patients (p=0.09). Notably, Black patients were 46% less likely to experience pCR than = White patients (RR: 0.54, 95% CI: 0.28-1.02) in the stage III group. Among patients less than 50 years of age, 41.18% of Black patients experienced pCR compared to 84.21% of White patients (p=0.01), where younger Black patients were 51% less likely to experience pCR than younger White patients (RR: 0.49, 95% CI: 0.27-0.89) There were no observed racial differences in pCR rates for stage I/II patients (p=1.00) or patients aged 50 years or older (p=0.64). Discussion These data demonstrate that Black women are at a pCR rate disadvantage receiving NACT/pembrolizumab. This is primarily driven by advanced stage and young age. Black patients with stage III cancers had significantly lower pCR rates than their White counterparts after having received the same treatment. Additionally, young patients who are Black have significantly lower pCR rates. For older patients and those with stage I/II disease, there was no statistically significant difference in pCR rates between Black vs White race. Conclusion While NACT/pembrolizumab offers overall increased rates of pCR, Black women, especially those who are young or with more advanced disease at presentation, still have worse outcomes. Further research is needed to determine underlying predictors or alternative treatments to benefit these populations. Table 1 Distribution of Pathological Complete Response Following NACT/pembrolizumab treatment (Nf91) Table 2 Crude Risk Ratios for the Relationship between Race and Pathological Complete Response by Age and Stage (Nf92) Citation Format: Melanie Sheen, Caitlin Taylor, Rabia Cattie, Melyssa Bratton, Meredith Lakey, Victoria Chung, Erin Biggs. Impact of age and stage on pathologic complete response rates in Black vs White patients with 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 PO5-03-07.

Abstract 2753: Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy

Abstract Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and has the worst survival rate. Immunotherapy is a new treatment option but resistance is frequent. We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein involved in metastasis (MTS) dissemination. Here, we evaluated the impact of the axis TNF/MUC4 on the invasive capacity of TNBC cell lines and in a preclinical model of spontaneous MTS. Finally, we assessed the clinical impact of MUC4 expression in TNBC patients. TNF blockade was achieved with etanercept (E), which blocks the soluble (sTNF) and transmembrane isoform of TNF, or with the dominant negative protein INB03 (DN) which neutralizes only sTNF. BT-549 and MDA-MB-231 human TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. To assess the impact of TNF blockade on tumor cells and its effect on the tumor microenvironment, we collected conditioned media (CM) from MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 and MDA-MB-231 was impaired with CM-Eta (p≤0.01) and CM-DN (p≤0.01 and p≤0.05, respectively). Female BALB/c mice bearing the TNBC LMM3 tumors were treated with IgG, DN, anti-PD-1 antibodies or DN+anti-PD-1 for two weeks. After the treatments we performed surgery to remove the primary tumor and the animals were sacrificed 2 weeks later. No effect on tumor growth was observed. However, the DN+anti-PD-1 group was the only one with less of 3 MTS per lung (p≤0.05). We obtained tumor extracts and found that the tumors of the animals treated with DN or DN+anti-PD-1 had decreased MUC4 expression measured by Western blot. We determined the presence of TILs by H &amp; E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 56 early TNBC patients. MUC4 expression was inversely correlated with TILs presence (p=0.0003). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM and MDA-MB-231-CM on T cell migration. CM-DN from both cell lines increased T cell migration compared to control CM (p≤0.01). MUC4 expression was inversely correlated with Ki67 (≥30%, p=0.036), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher MTS risk (p=0.005). MUC4 is associated with poorly-infiltrated TNBC, and sTNF blockade downregulates its expression decreasing MTS when combined with anti-PD-1. We propose the TNF as a new target for the treatment of TNBC, and MUC4 as a predictive biomarker to guide a combined treatment of TNF blockers with immunotherapy. Citation Format: Florencia Luciana Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, Maria Florencia Mercogliano, Roxana Schillaci. Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2753.

Abstract A050: CD8+ T cell exhaustion and their DNA damage in BRCA1 mutated TNBC

Abstract PARP Poly (ADP-ribose) polymerase inhibitors (PARPi) are treatment options in TNBC patients with inherited BRCA1 mutations. Recent studies have shown that immune checkpoint therapies (ICT) in combination with PARPi are effective in other solid cancer types such as melanoma, pancreatic and BRCA deficient ovarian cancer. In early and advanced BRCA1 TNBC, ICT studies are limited and not fully elucidated especially for new ICT that target LAG-3, CTLA-4, ICOS. There is a need for identifying potential prognostic markers suggesting which patients could benefit most from ICT. Therefore, we conducted a detailed single cell analysis of the tumor microenvironment (TME) mainly focused on immune checkpoint expression, CD8 T cell exhaustion and DNA damage associated with BRCA1 TNBC compared to WT TNBC. Combining single cell spatial tissue multiplexing (PhenoCycler) (BRCA1 TNBC n=77, WT TNBC n=55), scRNA seq (BRCA2 n=1, WT n=5), FLOW (PBMCs: BRCA1 n=13, BRCA2 n=17, WT n=62) as well as bulk RNA seq (BRCA1 TNBC n=35, WT TNBC n=11), we performed a detailed analysis of the TME associated with BRCA1 and WT breast cancer. We developed a 32-plex antibody panel that evaluated T-cells and other immune cells with markers of exhaustion, DNA damage and immune checkpoint expression. CD8+ T cell frequency was significantly increased in BRCA1 TNBC compared to WT TNBC (p=0.0015). A detailed CD8 T cell characterization revealed increased dysfunctional/exhausted cells (e.g., TOX+/LAG-3+/PD-1+), T cell DNA damage (CD3e+/CD8+/RAD51+, p=0.0047) and LAG-3 transcript levels in BRCA1 TNBC compared to WT TNBC (p=0.0625). Furthermore, immune checkpoint protein expression in tumor- (PD-L1) and immune cells (PD-1, LAG-3, ICOS) was increased in the TME associated with BRCA1 TNBC compared to WT TNBC. Of note, we also found that PBMCs of healthy tumor-free BRCA mutation carriers contained significantly increased CD8+/PD+1 T cells compared to WT (p=0.012). Our data indicates that BRCA1 haploinsufficiency leads to DNA damaged and dysfunctional CD8+ T cells within the TME compared to WT. Thus, the TME of BRCA1 TNBC patients is more immune suppressive due to increased exhausted CD8+ T cell frequency, their DNA damage as well as immune checkpoint expression of both tumor-/and immune cells compared to WT TNBC. The effect of BRCA1 haploinsufficiency on immune cells is important to consider when trials of ICT options are designed. Citation Format: Dana Pueschl, Anupma Nayak, Derek A. Oldridge, William Chandler, Bria Fulmer, Sokratis Apostolidis, Danielle Bragen, Bradley Wubbenhorst, John Pluta, kConFab Investigator, Antonis Antoniou, Georgia Chenevix-Trench, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. CD8+ T cell exhaustion and their DNA damage in BRCA1 mutated TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A050.

Abstract B033: Targeting CaMKK2 inhibits actin cytoskeletal assembly to suppress cancer metastasis

Abstract Triple-negative breast cancers (TNBCs) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Further, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. Citation Format: Debarati Mukherjee, Rebecca A Previs, Corinne N Haines, Muthana Al Abo, Patrick K Juras, Kyle C Strickland, Binita Chakraborty, Regina Whitaker, Steven R Patierno, Jennifer A Freedman, Ching-Yi Chang, Donald P McDonnell. Targeting CaMKK2 inhibits actin cytoskeletal assembly to suppress cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B033.

ASO Author Reflections: Immunotherapy and Breast Cancer-Spotlight on Early Stage TNBC.

ASO Author Reflections: Immunotherapy and Breast Cancer-Spotlight on Early Stage TNBC.

Differential benefit of capecitabine-based chemotherapy among TNBC subtypes in the context of the CBCSG010 study.

528 Background: Our previous study classified TNBCs into four mRNA subtypes, provided additional insights into TNBC heterogeneity and potential therapeutic options. Then we conducted a phase Ib/II subtyping-based and genomic biomarker-guided umbrella trial (FUTURE, NCT03805399 ), demonstrated the clinical benefit of subtyping-based targeted therapy for refractory metastatic TNBC. Here, we further evaluate the prognosis and predictive value of molecular classification in early TNBC in CBCSG010 trial (NCT01642771), which is a prospective, randomized phase III trial confirmed adding capecitabine to anthracycline-taxane-based adjuvant chemotherapy significantly improved survival in TNBC. Methods: Tumor tissues and pathological sections were retrospectively collected. Immunohistochemical (IHC) staining and hematoxylin and eosin (HE) staining were performed on paraffin-embedded sections to conduct TNBC IHC subtype staining. RNA-sequencing were performed to characterize the intrinsic molecular features of TNBC microenvironment. Results: 585 patients enrolled in the CBCSG010 trial, 450 patients had pathological sections, among which 207 patients achieved successfully stained sections. PDL1, CD8 and stromal tumor infiltrating lymphocytes (sTILs) were chosen for identified TNBC Immune enriched phenotype. Patients with ≥20% positive tumor cell proportion score (TPS) of PDL1, ≥10% positive cells of CD8, and ≥10% positive sTILs were significantly associated with better 5y-DFS (disease-free survival). Among Immune enriched TNBC, kaplan-Meier curves showed that DFS rates were 96.4% and 73.7% in the capecitabine and control groups. Transcriptome data was used to picture the immune microenvironment landscape, showed that “immune-hot” patients (immune cells or immune genes enriched) were more likely to benefit from capecitabine treatment. Conclusions: TNBC subtypes is significantly associated with DFS, with Immune enriched phenotype achieving the best outcome, and “immune-hot” patients are more likely to benefit from adjuvant capecitabine. Designing future clinical trials adding immunotherapy in adjuvant treatment of TNBC might base on molecular features.

Prognostic value of systemic inflammatory response in early-stage triple-negative breast cancer.

e14562 Background: Among breast cancer (BC) triple-negative (TN) subtype is the most immunogenic. Host and systemic immune response characterised by tumor-infiltrating lymphocytes (TILs) and immune cells in peripheral blood shape the net anti-tumor immune status. The systemic immune-inflammation index (SII) represents an effective indicator of the peripheral immune status. To date, TILs and SII have been largely examined separately. Therefore, in the present study, we investigated the prognostic value and relationship between pretreatment TILs and SII in a large number of unselected patients (pts) with early (e) stage TNBC. Methods: A total of 301 consecutive eTNBC pts treated with curative intent at Oncology Institute Ljubljana from January 2000 till August 2007 were investigated. Histopathological and clinical data were obtained retrospectively from the medical records. TILs were scored as intensive vs others. SII was determined by platelet × neutrophil/lymphocyte. Univariate and multivariate analyses were performed using the Cox proportional hazard model. The optimal cut-off value of SII was 675 and was evaluated using the receiver operating characteristic (ROC) curve. Results: The median follow-up period was 17,6 years (16.9-18.3), and the entire cohort`s 10-year DFS was 67.5% and 10-year OS 72.1%. The median age was 54 (53-57) years. Most pts were diagnosed node negative (40.7%), with larger tumors (≥ 2 cm, 57.1%), grade 3 (83.5%) and underwent perioperative chemotherapy (cht) (81%). Patients with positive lymph nodes (LN) had higher SII (r=0.21, p=0.003), no correlation between LN and TILs was found. Patients with higher TILs had significantly better OS (HR: 0.40, 95% CI 0.37-0.97, p=00.004), and lower SII predicted significantly better DFS and OS (HR for DFS: 1.65 95% CI 1,03-2.67, p=0.038, HR for OS: 1.72, 95% CI 1.11-2.65, p=0,015). In multivariate analysis, positive LN status retained its independent prognostic value for worse DFS and positive LN and higher SII for worse OS. Conclusions: Our data confirms important role of systemic immune response in the prognosis of eTNBC. The survival of these pts is significantly influenced by the pretreatment SII and TILs, the prognostic value of SII seems to be stronger in our cohort. However, positive lymph node status remains the strongest predictor for worse survival in eTNBC. [Table: see text]

Abstract 4600: Phenotypic &amp; functional diversity of tumor associated neutrophils in murine breast tumor models

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic &amp; functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

P125 Neoadjuvant pembrolizumab + chemotherapy vs placebo + chemotherapy followed by adjuvant pembrolizumab vs placebo for early TNBC: surgical outcomes from the phase 3 KEYNOTE-522 study

P125 Neoadjuvant pembrolizumab + chemotherapy vs placebo + chemotherapy followed by adjuvant pembrolizumab vs placebo for early TNBC: surgical outcomes from the phase 3 KEYNOTE-522 study

Abstract P2-21-09: Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Justin D. Wilson, Nika Shekastehband, Catherine HEDRICK, Alicia Arnold, Roni Bollag, Huidong Shi, Hasan Korkaya. Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice [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 P2-21-09.