Response In Triple-negative Breast Cancer Research Articles

Abstract C016: Age dictates immune response within the tumor microenvironment of triple negative breast cancer

Abstract Age is the biggest risk factor for developing most forms of cancer. In breast cancer, over 50% of patients are over 60 years old; however, less than 25% of patients enrolled in clinical trials are in this age bracket. Furthermore, most mouse modeling is performed in young mice, meaning that from preclinical through clinical stages patients ages are not accurately reflected in trials designs. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no targeted therapies, so many treatment strategies utilize chemotherapy in combination with immunotherapy – which relies on a functional immune response. It is well known that immune function declines as we age, and thus therapeutic responses may differ with age. It is yet unclear how the tumor immune microenvironment changes with age, and how this may impact therapeutic response. Here, we utilize a mouse model which mimics hallmarks of human immune aging to understand how age impacts TNBC immune responses to dictate disease progression and therapeutic response. Immunologically young (8 weeks old) and aged (∼12 months old) FVB mice were orthotopically injected with syngeneic Met1 TNBC cells. Once tumors became palpable, mice were treated with paclitaxel chemotherapy or vehicle control and/or anti-PDL1 immunotherapy to mimic first-line therapeutic strategies. Tumors grew significantly faster in young control mice compared to aged, but young mice had a more robust response to therapy. Notable, only young mice had a significant benefit of combo therapy over monotherapy alone. Despite aged mice having more circulating CD8+ and CD4+ T cells, tumors in aged mice had significantly more infiltration of CD8+ cytotoxic T cells and FOXP3+ inhibitory T-regulatory cells compared to young control mice, yet only tumors in young mice exhibited an increase in T cell infiltration with therapy. Additionally, the ratio of CD8+ T cells and FOXP3+ Tregs was unchanged with age or treatment. Depletion of either CD8+ or CD4+ T cells led to a complete loss of age-associated differences in tumor growth. RNA sequencing analysis suggests that infiltrating T cells in aged mice may be exhausted due to a chronic interferon (IFN) phenotype, indicated by enrichment of IFN-alpha, IFN-gamma, and IL2 pathways, as well as increased expression of immune exhaustion markers PD1, CTLA4, TIM3 and LAG3. Neutralization of IFN-gamma blocks therapeutic response to anti-PDL1 in young mice but has no impact on aged mice. Analysis of published single cell RNAseq data indicated the same IFN response pathways enriched with age in human TNBC. These results demonstrate that the aging tumor immune microenvironment significantly impacts tumor progression and therapeutic response and suggest that targeting IFN signaling may lead to improved therapeutic responses. Together these findings establish a significant impact of immune aging on TNBC disease progression and response to therapy, highlighting the importance of developing age- stratified treatment strategies to improve responses in patients of all ages. Citation Format: Milos Spasic, Adrienne Parsons, Abigail Recko, Elizabeth Mittendorf, Peter van Galen, Sandra McAllister. Age dictates immune response within the tumor microenvironment of triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C016.

The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

BackgroundBreast cancer, one of the most common forms of cancer, is associated with the highest cancer-related mortality among women worldwide. In comparison to other types of breast cancer, patients diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst outcome because current therapies do not produce long-lasting responses. Hence, innovative therapies that produce persisting responses are a critical need. We previously discovered that hyperactivating purinergic receptors (P2RXs) by increasing extracellular adenosine triphosphate (eATP) concentrations enhances TNBC cell lines’ response to chemotherapy. Heparan sulfate inhibits multiple extracellular ATPases, so it is a molecule of interest in this regard. In turn, heparanase degrades polysulfated polysaccharide heparan sulfate. Importantly, previous work suggests that breast cancer and other cancers express heparanase at high levels. Hence, as heparan sulfate can inhibit extracellular ATPases to facilitate eATP accumulation, it may intensify responses to chemotherapy. We postulated that heparanase inhibitors would exacerbate chemotherapy-induced decreases in TNBC cell viability by increasing heparan sulfate in the cellular microenvironment and hence, augmenting eATP.MethodsWe treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with paclitaxel (cytotoxic chemotherapeutic) with or without the heparanase inhibitor OGT 2115 and/or supplemental heparan sulfate. We evaluated cell viability and the release of eATP. Also, we compared the expression of heparanase protein in cell lines and tissues by immunoblot and immunohistochemistry, respectively. In addition, we examined breast-cancer-initiating cell populations using tumorsphere formation efficiency assays on treated cells.ResultsWe found that combining heparanase inhibitor OGT 2115 with chemotherapy decreased TNBC cell viability and tumorsphere formation through increases in eATP and activation of purinergic receptors as compared to TNBC cells treated with single-agent paclitaxel.ConclusionOur data shows that by preventing heparan sulfate breakdown, heparanase inhibitors make TNBC cells more susceptible to chemotherapy by enhancing eATP concentrations.

A computational analysis of the oncogenic and anti-tumor immunity role of P4HA3 in human cancers

Prolyl-4-hydroxylase subunit alpha3 (P4HA3) is a triple helical procollagen synthesis protein. The role of P4HA3 in cancer development is not well known and lacks comprehensive analyses among human cancers. This study aimed to investigate the relationship between P4HA3 expression and anti-tumor immunity and its prognostic value in pan-cancer. P4HA3 expression was analyzed from TIMER2.0, GTEx, GEPIA2.0 and TCGA databases. Genetic and DNA methylation alterations, survival analysis and proteins co-expression analysis of P4HA3 in cBio Cancer Genomics Portal, TCGA, GSCA and TIMER2.0. The correlation between P4HA3 expression and immune infiltration was analyzed by TIDE, XCELL, MCPCOUNTER, and EPIC. We performed EdU and transwell experiments to evaluate the influence of P4HA3 on the proliferation, migration and invasion abilities of different tumors. Patients derived xenograft (PDX) and subcutaneous transplantation models were utilized to explore the correlation between P4HA3 and immunotherapy response in triple-negative breast cancer (TNBC). Among 33 types of cancers, P4HA3 had generally different expression between different tumors, further analysis showed that the expression of P4HA3 was correlated with the cells infiltration of the tumor microenvironment (TME). The expression of P4HA3 was positively with the cell proliferation markers and epithelial-mesenchymal transition (EMT) markers. Moreover, P4HA3 deficiency inhibited the proliferation, migration and invasion abilities of tumor cells, and promoted anti-tumor immunotherapy of PD-1/PD-L1 inhibitor. This pan-cancer analysis of P4HA3 provides a comprehensive understanding of its oncogenic and prognosis role in different cancers, P4HA3 abnormal expression could be a useful biomarker for predicting the effectiveness of immunotherapy in cancer patients.

Integrated analysis reveals prognostic correlation and immune characteristics of a tumor-associated macrophage-based risk signature in triple-negative breast cancer.

Tumor-associated macrophages play a critical role in the progression and immune response of triple-negative breast cancer (TNBC). Our study aimed to explore the characteristics of tumor-associated macrophages (TAMs) in TNBC, construct a risk signature associated with TAM clusters, and verify its relationship with prognosis and immune-related characteristics. Firstly, we identified four TAM clusters and determined prognosis-related clusters in TNBC based on the single-cell RNA sequencing (scRNA-seq) data. Subsequently, the TAM-related prognostic genes were obtained by the univariate Cox regression analysis and an 8-gene risk signature was then constructed by least absolute shrinkage and selection operator (LASSO) regression based on these TAM-related prognostic genes. Analyses of immune characteristics showed a significant association between the signature with stromal and immune scores, as well as some immune cells. Multivariate analysis revealed that the risk signature was an independent prognostic factor for TNBC, and its value in predicting immunotherapeutic outcomes was also confirmed. A novel nomogram integrating the stage and TAM-based risk signature was constructed, which exhibited favorable predictability and reliability in the prognosis prediction of TNBC. Finally, the increasing expression of GPR34 which is one of the eight hub genes was explored in TNBC by experiments including reverse-transcriptase polymerase chain reaction, western blot, and immunohistochemistry. Our study may provide unique insights into obtaining independent prognostic factors, improving immunotherapeutic strategies, and identifying effective therapeutic targets for TNBC.

Caspase-1-dependent spatiality in triple-negative breast cancer and response to immunotherapy.

Tumor immune microenvironment (TIME) spatial organization predicts outcome and therapy response in triple-negative breast cancer (TNBC). An immunosuppressive TIME containing elevated tumor-associated macrophages (TAM) and scarce CD8+ T cells is associated with poor outcome, but the regulatory mechanisms are poorly understood. Here we show that ETS1-driven caspase-1 expression, required for IL1β processing and TAM recruitment, is negatively regulated by estrogen receptors alpha (ERα) and a defining feature of TNBC. Elevated tumoral caspase-1 is associated with a distinct TIME characterized by increased pro-tumoral TAMs and CD8+ T cell exclusion from tumor nests. Mouse models prove the functional importance of ERα, ETS1, caspase-1 and IL1β in TIME conformation. Caspase-1 inhibition induces an immunoreactive TIME and reverses resistance to immune checkpoint blockade, identifying a therapeutically targetable mechanism that governs TNBC spatial organization.

Factors Affecting the Response of Triple-Negative Breast Cancer (TNBC) to Neoadjuvant Chemotherapy: A Meta-Analysis

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. Neoadjuvant chemotherapy (NAC) is often used to downstage tumors before surgery, but response rates vary. This meta-analysis aims to identify factors that influence TNBC’s response to NAC. Methods: PubMed, Embase, and Cochrane Library databases were searched (2018-2024) for studies evaluating factors associated with TNBC response to NAC. Data on patient demographics, tumor characteristics, NAC regimens, and response rates were extracted. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using random-effects models. Results: Twenty-three studies (n=4,512 patients) were included. Younger age (OR 1.82, 95% CI 1.35-2.46), smaller tumor size (OR 0.58, 95% CI 0.41-0.82), lower clinical stage (OR 0.39, 95% CI 0.27-0.56), and absence of lymph node involvement (OR 0.42, 95% CI 0.31-0.57) were associated with improved pathological complete response (pCR) rates. NAC regimens containing platinum agents (OR 2.15, 95% CI 1.54-2.99) and immune checkpoint inhibitors (OR 1.78, 95% CI 1.23-2.58) also showed higher pCR rates. Conclusion: This meta-analysis identified several factors associated with improved TNBC response to NAC, including younger age, smaller tumor size, lower clinical stage, absence of lymph node involvement, and use of platinum-based or immunotherapy-containing NAC regimens. These findings can inform patient selection and treatment optimization for NAC in TNBC.

Tumour immune characterisation of primary triple-negative breast cancer using automated image quantification of immunohistochemistry-stained immune cells

The tumour immune microenvironment (TIME) in breast cancer is acknowledged with an increasing role in treatment response and prognosis. With a growing number of immune markers analysed, digital image analysis may facilitate broader TIME understanding, even in single-plex IHC data. To facilitate analyses of the latter an open-source image analysis pipeline, Tissue microarray MArker Quantification (TMArQ), was developed and applied to single-plex stainings for p53, CD3, CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 antibody) in a 218-patient triple negative breast cancer (TNBC) cohort with complementary pathology scorings, clinicopathological, whole genome sequencing, and RNA-sequencing data. TMArQ’s cell counts for analysed immune markers were on par with results from alternative methods and consistent with both estimates from human pathology review, different quantifications and classifications derived from RNA-sequencing as well as known prognostic patterns of immune response in TNBC. The digital cell counts demonstrated how immune markers are coexpressed in the TIME when considering TNBC molecular subtypes and DNA repair deficiency, and how combination of immune status with DNA repair deficiency status can improve the prognostic stratification in chemotherapy treated patients. These results underscore the value and potential of integrating TIME and specific tumour intrinsic alterations/phenotypes for the molecular understanding of TNBC.

Use of Pretreatment Perfusion MRI-based Intratumoral Heterogeneity to Predict Pathologic Response of Triple-Negative Breast Cancer to Neoadjuvant Chemoimmunotherapy.

Background Neoadjuvant chemoimmunotherapy (NACI) has significantly increased the rate of pathologic complete response (pCR) in patients with early-stage triple-negative breast cancer (TNBC), although predictors of response to this regimen have not been identified. Purpose To investigate pretreatment perfusion MRI-based radiomics as a predictive marker for pCR in patients with TNBC undergoing NACI. Materials and Methods This prospective study enrolled women with early-stage TNBC who underwent NACI at two different centers from August 2021 to July 2023. Pretreatment dynamic contrast-enhanced MRI scans obtained using scanners from multiple vendors were analyzed using the Tofts model to segment tumors and analyze pharmacokinetic parameters. Radiomics features were extracted from the rate constant for contrast agent plasma-to-interstitial transfer (or Ktrans), volume fraction of extravascular and extracellular space (Ve), and maximum contrast agent uptake rate (Slopemax) maps and analyzed using unsupervised correlation and least absolute shrinkage and selector operator, or LASSO, to develop a radiomics score. Score effectiveness was assessed using the area under the receiver operating characteristic curve (AUC), and multivariable logistic regression was used to develop a multimodal nomogram for enhanced prediction. The discrimination, calibration, and clinical utility of the nomogram were evaluated in an external test set. Results The training set included 112 female participants from center 1 (mean age, 52 years ± 11 [SD]), and the external test set included 83 female participants from center 2 (mean age, 47 years ± 11). The radiomics score demonstrated an AUC of 0.80 (95% CI: 0.70, 0.89) for predicting pCR. A nomogram incorporating the radiomics score, grade, and Ki-67 yielded an AUC of 0.86 (95% CI: 0.78, 0.94) in the test set. Associations were found between higher radiomics score (>0.25) and tumor size (P < .001), washout enhancement (P = .01), androgen receptor expression (P = .009), and programmed death ligand 1 expression (P = .01), demonstrating a correlation with tumor immune environment in participants with TNBC. Conclusion A radiomics score derived from pharmacokinetic parameters at pretreatment dynamic contrast-enhanced MRI exhibited good performance for predicting pCR in participants with TNBC undergoing NACI, and could potentially be used to enhance clinical decision making. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Rauch in this issue.

1948P Analysis of cytokines, chemokines, and tumor-infiltrating lymphocytes as immunological markers predicting pathological complete response in triple-negative breast cancer: Exploratory analysis of the NACATRINE trial

1948P Analysis of cytokines, chemokines, and tumor-infiltrating lymphocytes as immunological markers predicting pathological complete response in triple-negative breast cancer: Exploratory analysis of the NACATRINE trial

Predictive value of tumor-infiltrating lymphocytes for neoadjuvant therapy response in triple-negative breast cancer: A systematic review and meta-analysis.

The association between tumor-infiltrating lymphocyte (TIL) levels and the response to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC) remains unclear. To investigate the predictive potential of TIL levels for the response to NAT in TNBC patients. A systematic search of the National Center for Biotechnology Information PubMed database was performed to collect relevant published literature prior to August 31, 2023. The correlation between TIL levels and the NAT pathologic complete response (pCR) in TNBC patients was assessed using a systematic review and meta-analysis. Subgroup analysis, sensitivity analysis, and publication bias analysis were also conducted. A total of 32 studies were included in this meta-analysis. The overall meta-analysis results indicated that the pCR rate after NAT treatment in TNBC patients in the high TIL subgroup was significantly greater than that in patients in the low TIL subgroup (48.0% vs 27.7%) (risk ratio 2.01; 95% confidence interval 1.77-2.29; P < 0.001, I 2 = 56%). Subgroup analysis revealed that the between-study heterogeneity originated from differences in study design, TIL level cutoffs, and study populations. Publication bias could have existed in the included studies. The meta-analysis based on different NAT protocols revealed that all TNBC patients with high levels of TILs had a greater rate of pCR after NAT treatment in all protocols (all P ≤ 0.01), and there was no significant between-protocol difference in the statistics among the different NAT protocols (P = 0.29). Additionally, sensitivity analysis demonstrated that the overall results of the meta-analysis remained consistent when the included studies were individually excluded. TILs can serve as a predictor of the response to NAT treatment in TNBC patients. TNBC patients with high levels of TILs exhibit a greater NAT pCR rate than those with low levels of TILs, and this predictive capability is consistent across different NAT regimens.

Predicting the Prognosis and Immunotherapeutic Response of Triple-Negative Breast Cancer by Constructing a Prognostic Model Based on CD8+ T Cell-Related Immune Genes

ObjectiveTriple-negative breast cancer (TNBC) poses a significant challenge for treatment efficacy. CD8+ T cells, which are pivotal immune cells, can be effectively analyzed for differential gene expression across diverse cell populations owing to rapid advancements in sequencing technology. By leveraging these genes, our objective was to develop a prognostic model that accurately predicts the prognosis of patients with TNBC and their responsiveness to immunotherapy. MethodsSample information and clinical data of TNBC were sourced from The Cancer Genome Atlas and METABRIC databases. In the initial stage, we identified 67 differentially expressed genes associated with immune response in CD8+ T cells. Subsequently, we narrowed our focus to three key genes, namely CXCL13, GBP2, and GZMB, which were used to construct a prognostic model. The accuracy of the model was assessed using the validation set data and receiver operating characteristic (ROC) curves. Furthermore, we employed various methods, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, immune infiltration, and correlation analyses with CD274 (PD-L1) to explore the model's predictive efficacy in immunotherapeutic responses. Additionally, we investigated the potential underlying biological pathways that contribute to divergent treatment responses. ResultsWe successfully developed a model capable of predicting the prognosis of patients with TNBC. The areas under the curve (AUC) values for the 1-, 3-, and 5-year survival predictions were 0.618, 0.652, and 0.826, respectively. Employing this risk model, we stratified the samples into high- and low-risk groups. Through KEGG enrichment analysis, we observed that the high-risk group predominantly exhibited enrichment in metabolism-related pathways such as drug and chlorophyll metabolism, whereas the low-risk group demonstrated significant enrichment in cytokine pathways. Furthermore, immune landscape analysis revealed noteworthy variations between (PD-L1) expression and risk scores, indicating that our model effectively predicted the response of patients to immune-based treatments. ConclusionOur study demonstrates the potential of CXCL13, GBP2, and GZMB as prognostic indicators of clinical outcomes and immunotherapy responses in patients with TNBC. These findings provide valuable insights and novel avenues for developing immunotherapeutic approaches targeting TNBC.

Multiparametric MRI–based radiomic models for early prediction of response to neoadjuvant systemic therapy in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is often treated with neoadjuvant systemic therapy (NAST). We investigated if radiomic models based on multiparametric Magnetic Resonance Imaging (MRI) obtained early during NAST predict pathologic complete response (pCR). We included 163 patients with stage I-III TNBC with multiparametric MRI at baseline and after 2 (C2) and 4 cycles of NAST. Seventy-eight patients (48%) had pCR, and 85 (52%) had non-pCR. Thirty-six multivariate models combining radiomic features from dynamic contrast-enhanced MRI and diffusion-weighted imaging had an area under the receiver operating characteristics curve (AUC) > 0.7. The top-performing model combined 35 radiomic features of relative difference between C2 and baseline; had an AUC = 0.905 in the training and AUC = 0.802 in the testing set. There was high inter-reader agreement and very similar AUC values of the pCR prediction models for the 2 readers. Our data supports multiparametric MRI-based radiomic models for early prediction of NAST response in TNBC.

Neoadjuvant Chemotherapy Response in Triple-Negative Apocrine Carcinoma: Comparing Apocrine Morphology, Androgen Receptor, and Immune Phenotypes.

Apocrine differentiation and androgen receptor (AR) positivity represent a specific subset of triple-negative breast cancer (TNBC) and are often considered potential prognostic or predictive factors. To evaluate the response of TNBC to neoadjuvant chemotherapy (NAC) and to assess the impact of apocrine morphology, AR status, Ki-67 labeling index (Ki-67LI), and tumor-infiltrating lymphocytes (TILs). A total of 232 TNBC patients who underwent NAC followed by surgical resection in a single institute were analyzed. The study evaluated apocrine morphology and AR and Ki-67LI expression via immunohistochemistry from pre-NAC biopsy samples. Additionally, pre-NAC intratumoral TILs and stromal TILs (sTILs) were quantified from biopsies using a deep learning model. The response to NAC after surgery was assessed based on residual cancer burden. Both apocrine morphology and high AR expression correlated with lower Ki-67LI (P < .001 for both). Apocrine morphology was associated with lower postoperative pathologic complete response (pCR) rates after NAC (P = .02), but the difference in TILs between TNBC cases with and without apocrine morphology was not statistically significant (P = .09 for sTILs). In contrast, AR expression did not significantly affect pCR (P = .13). Pre-NAC TILs strongly correlated with postoperative pCR in TNBCs without apocrine morphology (P < .001 for sTILs), whereas TNBC with apocrine morphology demonstrated an indeterminate trend (P = .82 for sTILs). Although TIL counts did not vary significantly based on apocrine morphology, apocrine morphology itself was a more reliable predictor of NAC response than AR expression. Consequently, although apocrine morphology is a rare subtype of TNBC, its identification is clinically important.

TNBC response to paclitaxel phenocopies interferon response which reveals cell cycle-associated resistance mechanisms.

Paclitaxel is a standard of care neoadjuvant therapy for patients with triple negative breast cancer (TNBC); however, it shows limited benefit for locally advanced or metastatic disease. Here we used a coordinated experimental-computational approach to explore the influence of paclitaxel on the cellular and molecular responses of TNBC cells. We found that escalating doses of paclitaxel resulted in multinucleation, promotion of senescence, and initiation of DNA damage induced apoptosis. Single-cell RNA sequencing (scRNA-seq) of TNBC cells after paclitaxel treatment revealed upregulation of innate immune programs canonically associated with interferon response and downregulation of cell cycle progression programs. Systematic exploration of transcriptional responses to paclitaxel and cancer-associated microenvironmental factors revealed common gene programs induced by paclitaxel, IFNB, and IFNG. Transcription factor (TF) enrichment analysis identified 13 TFs that were both enriched based on activity of downstream targets and also significantly upregulated after paclitaxel treatment. Functional assessment with siRNA knockdown confirmed that the TFs FOSL1, NFE2L2 and ELF3 mediate cellular proliferation and also regulate nuclear structure. We further explored the influence of these TFs on paclitaxel-induced cell cycle behavior via live cell imaging, which revealed altered progression rates through G1, S/G2 and M phases. We found that ELF3 knockdown synergized with paclitaxel treatment to lock cells in a G1 state and prevent cell cycle progression. Analysis of publicly available breast cancer patient data showed that high ELF3 expression was associated with poor prognosis and enrichment programs associated with cell cycle progression. Together these analyses disentangle the diverse aspects of paclitaxel response and identify ELF3 upregulation as a putative biomarker of paclitaxel resistance in TNBC.

Ibulocydine Inhibits Migration and Invasion of TNBC Cells via MMP-9 Regulation.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer types, indicating a poor survival prognosis with a more aggressive biology of metastasis to the lung and a short response duration to available therapies. Ibulocydine (IB) is a novel (cyclin-dependent kinase) CDK7/9 inhibitor prodrug displaying potent anti-cancer effects against various cancer cell types. We performed in vitro and in vivo experiments to determine whether IB inhibits metastasis and eventually overcomes the poor drug response in TNBC. The result showed that IB inhibited the growth of TNBC cells by inducing caspase-mediated apoptosis and blocking metastasis by reducing MMP-9 expression in vitro. Concurrently, in vivo experiments using the metastasis model showed that IB inhibited metastasis of MDA-MB-231-Luc cells to the lung. Collectively, these results demonstrate that IB inhibited the growth of TNBC cells and blocked metastasis by regulating MMP-9 expression, suggesting a novel therapeutic agent for metastatic TNBC.

NeoTRACT: Phase II trial of neoadjuvant tumor infiltrating lymphocyte- and response-adapted chemoimmunotherapy for triple-negative breast cancer (TNBC).

TPS629 Background: Neoadjuvant chemoimmunotherapy is recommended for patients with high risk early stage TNBC. The 5 drug KEYNOTE-522 neoadjuvant chemoimmunotherapy regimen involves 5-6 months of treatment and is associated with suboptimal treatment completion due to toxicity. Approaches to deescalate chemoimmunotherapy without compromising outcomes for patients with highly chemosensitive disease are needed. Stromal tumor infiltrating lymphocyte (sTIL) enrichment is positively associated with pathologic complete response (pCR) in TNBC and may be useful to identify patients for whom de-escalation of neoadjuvant chemoimmunotherapy might be acceptable. NeoTRACT trial investigates deescalating neoadjuvant chemoimmunotherapy based on degree of pretreatment sTIL enrichment. We hypothesize that baseline immune upregulation assessed by sTILs can guide neoadjuvant treatment optimization in TNBC patients. Specifically, in patients with immune enriched TNBC (sTILs ≥30%), short anthracycline free taxane–platinum chemoimmunotherapy will yield high pCR rate. Methods: NeoTRACT is an ongoing open label phase II trial for patients with previously untreated TNM stage I (T&gt;1cm)–III TNBC. TNBC is defined as estrogen and progesterone receptor ≤10% and HER2 negative per ASCO/CAP criteria. Patients with T4 or N3 breast cancer are excluded. Patients must have adequate organ function and no contraindication to pembrolizumab. All participants initiate carboplatin (AUC 6) + docetaxel (75mg/m2) + pembrolizumab (200mg) (CbD+P) every 21 days. After registration, sTILs are evaluated centrally on pretreatment H&amp;E core needle biopsy slide using standard criteria. Participants are assigned to three neoadjuvant treatment regimens (A, B, or C) based on sTIL enrichment. Regimen A (sTILs ≥30%) includes four cycles CbD+P. All participants assigned to regimen A undergo breast MRI after four cycles: those with complete MRI response proceed to breast surgery; those with less than complete MRI response receive two additional cycles of CbD+P. Regimen B (sTILs 5-29%) includes six cycles of CbD+P. Regimen C (sTILs &lt;5%) includes four cycles of CbD+P followed by four cycles of doxorubicin (60mg/m2) + cyclophosphamide (600mg/m2) + P (200mg) (AC+P). All patients undergo surgery after completing the assigned neoadjuvant regimen. Breast/axillary surgery, adjuvant systemic therapy, and radiotherapy are recommended per standard of care. pCR is defined as absence of residual invasive disease in breast and axilla. Primary endpoint is pCR rate among participants with ≥30% sTILs. Secondary endpoints include residual cancer burden (RCB) rates in patients with ≥30% sTILs, pCR and RCB rates in patients with 5-29% and &lt;5% sTILs, and event free and overall survival for all patients. As of January 2024, 45 of 139 participants have been enrolled. Clinical trial information: NCT05645380 .

Co-assembly of cisplatin and dasatinib in hyaluronan nanogel to combat triple negative breast cancer with reduced side effects

Treatment for triple negative breast cancer (TNBC) remains a huge challenge due to the lack of targeted therapeutics and tumor heterogenicity. Cisplatin (Cis) have demonstrated favorable therapeutic response in TNBC and thus is used together with various kinase inhibitors to fight the heterogenicity of TNBC. The combination of Cis with SRC inhibitor dasatinib (DAS) has shown encouraging anti-TNBC efficacy although the additive toxicity was commonly observed. To overcome the severe side effects of this Cis involved therapy, here we co-encapsulated Cis and DAS into a self-assembled hyaluronan (HA) nanogel (designated as HA/Cis/DAS (HCD) nanogel) to afford the TNBC targeted delivery by using the 4T1 mouse model. The acquired HCD nanogel was around 181 nm in aqueous solution, demonstrating the pharmacological activities of both Cis and DAS. Taking advantages of HA's targeting capability towards CD44 that is overexpressed on many TNBC cells, the HCD could well maintain the anticancer efficacy of the Cis and DAS combination, significantly increase the maximum tolerated dose and relieve the renal toxicity in vivo. The current HCD nanogel provides a potent strategy to improve the therapeutic outcome of Cis and DAS combination and thus representing a new targeted treatment option for TNBC.

Abstract PO3-07-10: Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC

Abstract Background: Tumor immunology and immunosuppression has been observed to drive changes in intratumoral response to cytotoxic and immunotherapy in triple negative breast cancer (TNBC). While radiation therapy is standard of care for TNBC, there is a lack of research into the relationship between long term treatment response and CD8 immune infiltration in the context of a radiation resistant model. Positron emission tomography (PET) imaging allows for noninvasive monitoring of the tumor microenvironment that can precede changes in tumor volume, including approaches that allow for immune imaging of CD8 T-cell trafficking. Noninvasive PET imaging of radiation response has the potential to identify windows of enhanced response to secondary therapy and can guide interventional therapy. The goal of this study is to understand how radiation can prime the tumor microenvironment to be combined with other therapeutics in TNBC through changes in CD8 immune infiltration with [89Zr]-CD8 ImmunoPET imaging. Methods: Syngeneic parental 4T1 (radiation sensitive) and a developed radiation-resistant 4T1 sub-cell line were orthotopically injected into BALB/c mice (N = 5 control, N = 9-10 radiation treated for each model). After reaching a tumor volume of ~100 mm3, mice began treatment with 2 Gy fractionated radiation daily from day 0-5. On day 6, mice were injected with ~50 µCi of [89Zr]-CD8 minibody (IAB42) and imaged 24 hours post injection. The mean standardized uptake value (SUV) was quantified and normalized to heart SUV to extract out a tumor:heart SUVratio that describe the CD8 infiltration within tumors. Following imaging, tumors were either excised immediately for immunofluorescence against CD8 or monitored for longitudinal changes in tumor volume. A non-parametric T-test was used to assess for significance between groups. Results: In radiation sensitive 4T1 tumors, [89Zr]-CD8 ImmunoPET imaging indicated a 23% increase in CD8 immune infiltration, in radiation-treated tumors compared to control tumors on day 7 (p = 0.02). These immune alterations occurred prior to any changes in tumor volume (p = 0.63). Longitudinally, radiation treated tumors exhibited significant changes in tumor volume beginning on day 20 (p = 0.02), which is sustained until study endpoint on day 41 (p &amp;lt; 0.01). In radiation resistant 4T1 tumors, no significant change was observed in short term CD8 immune infiltration (p = 0.43) or in longitudinal tumor volume (p = 0.31) in response to radiation therapy, when compared to untreated tumors. Conclusions: [89Zr]-CD8 ImmunoPET imaging reveals significant increases in CD8 immune infiltration that is predictive of eventual response to radiation therapy in radiation sensitive models of TNBC. [89Zr]-CD8 PET imaging of radiation therapy response has the potential to increase the efficacy of precision medicine and prime the tumor microenvironment for secondary therapeutics, thereby improving tumor kill and reducing patient toxicity. Citation Format: Patrick Song, Shannon Lynch, Chloe DeMellier, Alessandro Mascioni, Fang Jia, Anna Sorace. Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC [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 PO3-07-10.

Abstract PO3-25-05: Eribulin Induces Chromosomal Instability and Enhances cGAS Expression in the Nucleus of Triple-Negative Breast Cancer

Abstract Background: Eribuliln (ERI), a microtubule polymelization, is approved for locally advanced or metastatic breast cancer. However, its effects on drug-naive cancers are not well understood. In EMBRACE trial, ERI improves overall survival (OS) of the patients with metastatic breast cancer. This result might suggest that ERI could be involved in the immune system. In particular, there are a few reports on the effects of ERI on the innate immune system. This study aimed to investigate how ERI influences the innate immune system, focusing on the cyclic-GMP-AMP synthase (cGAS), a DNA sensor that triggers the production of type-I interferons. Methods: Clinical samples from the JONIE-3 trial were analyzed using immunohistochemistry (IHC). 121 patients were assigned to 2 different neoadjuvant chemotherapy (NAC) groups receive ERI (Group E) or paclitaxel (Group P) followed by FEC. The patients of both groups were performed biopsy before and after chemotherapy. We performed IHC on 56 samples and examined for association with pathological complete response (pCR), which was defined as no invasive redidual tumor tissue in the breast. Additionally, 5 different cell lines were established to evaluate the acute and chronic effects of ERI treatment. The cell lines were as follows: no treatment (control), PTX for short time (PTX short), PTX for long time (PTX long), ERI for short time (ERI short) and ERI for long time (ERI long). We evaluated the acute effects of short-term dosing, while the chronic effects of long-term dosing, which mimic resistance to treatment. Protein expression of the cGAS-STING pathway was examined, along with cGAS and IFNβ expression levels and their impact on cell division in the above cell lines. Then at the cellular level, each cell lines were evaluated for differences in cGAS and IFNβ expression and their effects on cell division. The differences in cGAS expression between cytoplasmic and nuclear fractions were verified by the cell fractuation assay. In addition, mitotic abnormalities and cell proliferation were also assessed. Results: In the clinical trial, ERI did not significantly differ from paclitaxel in terms of pathological complete response (pCR). However, high cGAS expression in Group E (ERI) correlated with increased pCR rates, while no such correlation was observed in Group P (PTX) (Figure). Additionally, High IFNβ expression in Group E also correlated with increased pCR rates, differed from Group P. In vitro, ERI upregulated cGAS, STING, pIRF3, and IFNβ protein expression compared to PTX. Notably, ERI induced elevated cGAS expression in the nucleus, as confirmed by immunofluorescence and cell fracturation assays. Additionally, PTX and ERI differed in their ability to cause mitotic abnormalities. PTX induced more micronuclei cells than ERI, on the other hand ERI induced more micronuclei cells and mitotic slippage. These results were also verified by live cell imaging. Finally, the knockdown of cGAS resulted in accelerated cell proliferation. Conclusion: ERI promoted chromosomal instability, leading to increased cGAS expression, particularly in the nucleus. These findings contribute to our understanding of ERI's effects on the innate immune response in triple-negative breast cancer, potentially paving the way for improved therapeutic strategies. The differences in intensity scores between GroupP and GroupE in the immunostaining of cGAS High cGAS expression in Group E correlated with increased pCR rates (p=0.0375), while no such correlation was observed in Group P (p=0.2983). Citation Format: Hideyuki Yamada, Mamoru Takada, Aussie Suzuki, Yu Muhan, Takeshi Nagashima, Hiroshi Fujimoto, Junta Sakakibara, Masaharu Kasuya, Takahiko Kawate, Daishu Miura, Masato Suzuki, Masaru Miyashita, Kazutaka Narui, Yoshie Hasegawa, Takashi Ishikawa, Masayuki Otsuka. Eribulin Induces Chromosomal Instability and Enhances cGAS Expression in the Nucleus of 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 PO3-25-05.

Abstract PO5-03-06: Optimizing therapeutic regimens via digital twins to improve triple negative breast cancer response to neoadjuvant therapy

Abstract Introduction: Neoadjuvant systemic therapy (NAT) has been the standard-of-care of stage II-III, locally advanced triple-negative breast cancer (TNBC). However, about 50% of TNBC patients achieve a pathological complete response (pCR) to conventional neoadjuvant chemotherapy (NAC)1. Recently approved combination of NAC with the immunotherapy pembrolizumab, has improved the pCR rate by 7.5%2, although with a 44% risk of immune-related adverse reaction3. Aside from the need to develop new therapies with higher efficacy and lower toxicity, a critical barrier to improving TNBC response is the lack of rigorous ways to personally tailor therapeutic regimens. We seek to address this challenge by employing digital twins (i.e., mathematical models that provide virtual representation of individual patients and predict the changes at future time points) to systematically evaluate individual TNBC patient’s response to different NAT regimens, thereby patient-specifically optimizing treatments. Methods: A TNBC cohort (n = 139) from the ARTEMIS trial (NCT02276433)4 was used for this study. All patients received 4 cycles of Adriamycin/Cytoxan (A/C) every 2 weeks, followed by 12 cycles of weekly Taxol or experimental therapy in Phase II trials. All patients had surgery after NAT and post-surgical pathology to assess response status. For each patient, longitudinal MRIs were collected before, during, and after A/C. We have developed digital twins to integrate the longitudinal MRIs with a mechanism-based model to accurately predict TNBC response5. The model was based on a reaction-diffusion equation that describes the change in tumor cellularity due to migration, proliferation, and drug-induced death. With parameters personalized using MRIs, the patient-specific model (i.e., digital twin) significantly improved the accuracy to predict pCR5. We investigated patient-specific treatment optimization on 37 (19 pCR, 18 non-pCR) chemo-sensitive patients (≥ 70% volume decrease after A/C) who received only NAC. We evaluated the effect of altering the A/C/Taxol schedules on patient response. Specifically, each patient’s digital twin was used to predict the patient’s response to 128 clinically reasonable schedules of A/C/Taxol; i.e., 8 candidate A/C schedules combining with 16 candidate Taxol schedules (Table 1). The predicted response (pCR or non-pCR) from each alternative schedule was compared to the patient response from the actual treatment. Results: Without changing the total dose, shortening the duration of A/C/Taxol administration increased the treatment efficacy. The effectiveness of altering the schedules varied substantially in different patients. In particular, 8 patients who had non-pCR responses to their actual treatment were predicted to achieve pCR with the dense-dose Taxol (i.e., 4 cycles Taxol, 2 weeks per cycle), indicating a 21.62% improvement of pCR rate in the cohort. Discussion and conclusion: The preliminary results with our digital twin approach provided a unique opportunity of improving TNBC response to NAT through patient-specific optimization of therapeutic schedules. The ongoing effort focuses on accounting for toxicity and investigating the effects of altering therapy types and doses in combination with the schedules on the patient response. [1] Spring et al., Clin Cancer Res, 2020. [2] Schmid et al., NEJM, 2022. [3] Shah et al., Clin Cancer Res, 2022. [4] Yam et al., Clin Cancer Res, 2021. [5] Wu et al., Cancer Res, 2022. Table 1. Candidate therapeutic schedules Citation Format: Chengyue Wu, Ernesto Lima, Casey Stowers, Zhan Xu, Clinton Yam, Jong Bum Son, Jingfei Ma, Gaiane Rauch, Thomas Yankeelov. Optimizing therapeutic regimens via digital twins to improve triple negative breast cancer response to neoadjuvant therapy [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 PO5-03-06.