Triple-negative Breast Cancer Molecular Subtypes Research Articles

Evaluation of alternative prognostic thresholds for SP142 and 22C3 immunohistochemical PD-L1 expression in triple-negative breast cancer: results from a population-based cohort.

Immune checkpoint inhibitors are now a part of the treatment arsenal for triple-negative breast cancer (TNBC) but refinement of PD-L1 as a prognostic and predictive biomarker is a clinical priority. We aimed to evaluate the relevance of novel PD-L1 immunohistochemical (IHC) thresholds in TNBC with regard to PD-L1 gene expression, prognostic value, tumor infiltrating lymphocytes (TILs), and TNBC molecular subtypes. PD-L1 was scored in a tissue microarray with the SP142 (immune cell (IC) score) and the 22C3 (combined positive score; CPS) IHC assays and TIL abundance evaluated in whole slides in a population-based cohort of 237 early-stage TNBC patients. Survival analysis was performed and RNA sequencing data employed for molecular profiling. As expected, PD-L1 positivity (IC ≥ 1% and/or CPS ≥ 1) was significantly associated with better prognosis compared to zero PD-L1 expression. Importantly however, also patients with intermediate expression (IC > 0%, < 1%; CPS > 0, < 1) showed a trend toward improved outcome. Tumors with intermediate PD-L1 IHC expression also had intermediate PD-L1 (CD274) gene expression (mRNA). Patients who were both low in TILs (< 30%) and PD-L1 (IC < 1%; CPS < 1) tended to have the poorest prognosis. PD-L1 positive tumors clustered significantly more often as Immunomodulatory-high and Basal-Like 1-high TNBC molecular subtypes and were enriched in immune response and cell cycle/proliferation signaling pathways. PD-L1-zero tumors on the other hand were enriched in cell growth, differentiation, and metastatic potential pathways and clustered more prevalently as Luminal-Androgen-Receptor-high and Mesenchymal-high. PD-L1-intermediate tumors categorized with neither PD-L1-positive nor PD-L1-zero tumors on the hierarchical clustering level, consigning them as a unique subgroup. With both SP142 and 22C3, we identified an intermediate IHC PD-L1 group within TNBCs that was supported on the molecular level. Any PD-L1 IHC expression, even though it is < 1, tended to have positive prognostic impact. We suggest that the generally accepted threshold of PD-L1 IHC positivity in TNBC should be investigated further. The Swedish Cancerome Analysis Network - Breast (SCAN-B) study was retrospectively registered 2nd Dec 2014 at ClinicalTrials.gov; ID NCT02306096.

Advances in the Understanding of the Pathogenesis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by high aggressiveness, high malignancy, and poor prognosis compared to other breast cancer subtypes. This review aims to explore recent advances in understanding TNBC and to provide new insights and potential references for clinical treatment. We examined current literature on TNBC to analyze molecular subtypes, genetic mutations, signaling pathways, mechanisms of drug resistance, and emerging therapies. Findings highlight key aspects of TNBC's molecular subtypes, relevant mutations, and pathways, alongside emerging treatments that target drug resistance mechanisms. These insights into TNBC pathogenesis may help guide future therapeutic strategies and improve clinical outcomes for patients with 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.

Mesenchymal-like immune-altered is the fourth robust triple-negative breast cancer molecular subtype.

Robust molecular subtyping of triple-negative breast cancer (TNBC) is a prerequisite for the success of precision medicine. Today, there is a clear consensus on three TNBC molecular subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA), and basal-like immune-suppressed (BLIS). However, the debate about the robustness of other subtypes is still open. An unprecedented number (n = 1942) of TNBC patient data was collected. Microarray- and RNAseq-based cohorts were independently investigated. Unsupervised analyses were conducted using k-means consensus clustering. Clusters of patients were then functionally annotated using different approaches. Prediction of response to chemotherapy and targeted therapies, immune checkpoint blockade, and radiotherapy were also screened for each TNBC subtype. Four TNBC subtypes were identified in the cohort: LAR (19.36%); mesenchymal stem-like (MSL/MES) (17.35%); BLIA (31.06%); and BLIS (32.23%). Regarding the MSL/MES subtype, we suggest renaming it to mesenchymal-like immune-altered (MLIA) to emphasize its specific histological background and nature of immune response. Treatment response prediction results show, among other things, that despite immune activation, immune checkpoint blockade is probably less or completely ineffective in MLIA, possibly caused by mesenchymal background and/or an enrichment in dysfunctional cytotoxic T lymphocytes. TNBC subtyping results were included in the bc-GenExMiner v5.0 webtool ( http://bcgenex.ico.unicancer.fr ). The mesenchymal TNBC subtype is characterized by an exhausted and altered immune response, and resistance to immune checkpoint inhibitors. Consensus for molecular classification of TNBC subtyping and prediction of cancer treatment responses helps usher in the era of precision medicine for TNBC patients.

Triple Negative Breast Cancer: Molecular Subtype-Specific Immune Landscapes with Therapeutic Implications.

Triple Negative Breast Cancer (TNBC) is characterized by distinct molecular subtypes with unique biological and clinical features. This systematic review aimed to identify articles examining the differences in the tumor immune microenvironment (TIME) across different TNBC molecular subtypes. Six studies meeting inclusion criteria were analyzed, utilizing gene expression profiling and bioinformatic analyses to classify TNBC samples into molecular subtypes, as well as immunohistochemistry and cell deconvolution methods to characterize the TIME. Results revealed significant heterogeneity in immune cell composition among TNBC subtypes, with the immunomodulatory (IM) subtype demonstrating robust immune infiltration, composed mainly of adaptive immune cells along with an increased density of CTLA-4+ and PD-1+ TILs, high PD-L1 tumor cell expression, and upregulation of FOXP3+ Tregs. A more immunosuppressive TIME with a predominance of innate immune cells and lower levels of tumor-infiltrating lymphocytes (TILs) was observed in luminal androgen receptor (LAR) tumors. In mesenchymal stem-like (MSL) tumors, the TIME was mainly composed of innate immune cells, with a high number of M2 tumor-associated macrophages (TAMs), while the BL and M tumors displayed poor adaptive and innate immune responses, indicating an "immune-cold" phenotype. Differential activation of signaling pathways, genomic diversity, and metabolic reprogramming were identified as contributors to TIME heterogeneity. Understanding this interplay is crucial for tailoring therapeutic strategies, especially regarding immunotherapy.

Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer

Triple-negative breast cancer (TNBC) has high heterogeneity, poor prognosis, and limited treatment success. Recently, an immunohistochemistry-based surrogate classification for the “Fudan University Shanghai Cancer Center (FUSCC) subtyping” has been developed and is considered more suitable for clinical application. Seventy-one paraffin-embedded sections of surgically resected TNBC were classified into four molecular subtypes using the IHC-based surrogate classification. Genomic analysis was performed by targeted next-generation sequencing and the specificity of the subtypes was explored by bioinformatics, including survival analysis, multivariate Cox regression, pathway enrichment, Pyclone analysis, mutational signature analysis and PHIAL analysis. AKT1 and BRCA1 mutations were identified as independent prognostic factors in TNBC. TNBC molecular subtypes encompass distinct genomic landscapes that show specific heterogeneities. The luminal androgen receptor (LAR) subtype was associated with mutations in PIK3CA and PI3K pathways, which are potentially sensitive to PI3K pathway inhibitors. The basal-like immune-suppressed (BLIS) subtype was characterized by high genomic instability and the specific possession of signature 19 while patients in the immunomodulatory (IM) subtype belonged to the PD-L1 ≥ 1% subgroup with enrichment in Notch signaling, suggesting a possible benefit of immune checkpoint inhibitors and Notch inhibitors. Moreover, mesenchymal-like (MES) tumors displayed enrichment in the receptor tyrosine kinase (RTK)-RAS pathway and potential sensitivity to RTK pathway inhibitors. The findings suggest potential treatment targets and prognostic factors, indicating the possibility of TNBC stratified therapy in the future.

A 10-Gene Signature to Predict the Prognosis of Early-Stage Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a particularly challenging subtype of breast cancer, with a poorer prognosis compared to other subtypes. Unfortunately, unlike luminal-type cancers, there is no validated biomarker to predict the prognosis of patients with early-stage TNBC. Accurate biomarkers are needed to establish effective therapeutic strategies. In this study, we analyzed gene expression profiles of tumor samples from 184 TNBC patients (training cohort, n=76; validation cohort, n=108) using RNA sequencing. By combining weighted gene expression, we identified a 10-gene signature (DGKH, GADD45B, KLF7, LYST, NR6A1, PYCARD, ROBO1, SLC22A20P, SLC24A3, and SLC45A4) that stratified patients by risk score with high sensitivity (92.31%), specificity (92.06%), and accuracy (92.11%) for invasive disease-free survival. The 10-gene signature was validated in a separate institution cohort and supported by meta-analysis for biological relevance to well-known driving pathways in TNBC. Furthermore, the 10-gene signature was the only independent factor for invasive disease-free survival in multivariate analysis when compared to other potential biomarkers of TNBC molecular subtypes and T-cell receptor β diversity. 10-gene signature also further categorized patients classified as molecular subtypes according to risk scores. Our novel findings may help address the prognostic challenges in TNBC and the 10-gene signature could serve as a novel biomarker for risk-based patient care.

Abstract PO1-15-04: Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations

Abstract Introduction: While triple-negative breast cancer (TNBC) molecular subtypes have been associated with biological differences and clinical outcomes, studies have overwhelmingly been conducted in populations of European or Asian ancestry. Data collected across diverse populations is required to better leverage clinical directions from translational studies in TNBC. Through females recruited through the Black Women: Etiology and Survival of Triple-Negative Breast Cancers (BEST) study, we sought to characterize subtypes and explore associations. Methods: Our study included Black women diagnosed with early-stage (I-III) invasive TNBC at &amp;lt; = age 50 from 2005 to 2016, with recruitment from Florida and Tennessee state cancer registries. Germline DNA, tumor RNA, clinical outcomes, and risk factor data were collected. TNBC status in the BEST study was based on immunohistochemistry from pathology reports, cancer registry data, and self-reported data. Analyses on banked tumor samples, extracted at multiple time points, were conducted through PAM50 (Nanostring) and whole-transcriptome RNA-seq. TNBC subtype was assessed using TNBCtype-4 (Lehmann et al. 2016). Sources of batch effect were evaluated using PCA and corrected via limma. Genetic ancestry based on OncoArray or MEGA genotyping data was inferred using 1000Genomes continental reference samples. Immune cell characterization was performed via CIBERSORT and ESTIMATE. Statistical significance in comparing differences across subtypes was assessed via Chi-square test. Kaplan-Meier estimates and log-rank test were used for survival analysis. Results: RNA-seq from 114 self-reported Black females with TNBC was analyzed. TNBC subtype distribution in the BEST study cohort included 31.6% basal-like 1 (BL1), 28.9% basal-like 2 (BL2), 16.7% luminal androgen receptor (LAR), and 21.9% mesenchymal (M). While results were largely consistent with prior studies in patients of European or East Asian ancestry, a higher percentage of BL2 subtype was observed (28.9% vs. 21.0%, p=0.053). Based on PAM50 subtyping, 100% of tumors with BL1 and M were basal, whereas 68.8% of LAR and 86.7% of BL2 subtypes were basal. Tumors without subtype decreased from 9.8% to 1.1% after batch correcting for location at which RNA was extracted (Moffitt vs. Vanderbilt) and time between RNA banking and sequencing. All TNBC subtypes in this population demonstrated monocyte/activated dendritic cell predominance, with no significant subtype-specific associations with immune cell patterns. A lower relative proportion of M subtype was found in tumors from BRCA1/2 carriers, but this was not statistically significant due to the small number of carriers (8% vs. 18%; p=0.22). With a median follow-up time of 9 years, there was no significant difference in 10-year overall survival by TNBC subtype (p=0.18). However, patients with M subtype appeared to have worse survival relative to other subtypes combined (p=0.036). Conclusion: Our study is among the largest to date that interrogates TNBC subtypes and associated molecular/clinical data in self-reported Black females with invasive breast cancer. Our findings suggest that established TNBC subtyping can be applied in patients of African ancestry. Furthermore, data to correct for lab-based confounders remains critical; in this study, it enabled us to use many samples that could not be subtyped initially. Several population-specific patterns were observed, including no difference in 10-year overall survival across TNBC subtypes (consistent with prior data), but worse initial outcomes in M subtype; a preliminary association between BRCA1/2 carriers and non-M subtypes (in contrast to prior data); and no significant difference in immune cell distributions (in contrast to prior data mainly from patients of European ancestry). Studies of associations between TNBC subtypes, additional clinical data, and treatment data (type and response rates) are ongoing. Citation Format: Padma Rajagopal, Sonya Reid, Run Fan, Lindsay Venton, Anne Weidner, Mya Roberson, Susan Vadaparampil, Xuefeng Wang, Sean Yoder, Marilin Rosa, Jibril Hirbo, Jennifer Whisenant, Jennifer Pietenpol, Fei Ye, Tuya Pal, Brian Lehmann. Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-04.

Abstract PO2-15-05: Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of primary BC. This heterogeneous disease is the most aggressive BC subtype. TNBC is characterized by young age at onset, high-grade tumors, high risk of early recurrence and poor prognosis. Until recently, TNBC patients were considered as one group and all patients were treated with conventional chemotherapy. With the emergence of new therapies targeting distinct biological pathways, personalized medicine is now accessible by defining finer TNBC subtypes, e.g. molecular subtypes, and identifying their specific targetable pathways. Several studies based on transcriptomics have identified three to six TNBC molecular subtypes. Today, there is a clear consensus on three TNBC robust subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA) and basal-like immune-suppressed (BLIS). The debate about the robustness of other subtypes is still open, notably mesenchymal-like (M) and mesenchymal stem-like (MSL or MES). The goal of this study was to establish a robust molecular classification of TNBC that allow to determine the best treatment options. To optimize this process, we harvested as much transcriptomic data as possible. Available clinicopathological data linked to these data were also imported. To our knowledge, this is the largest study aimed at subtyping TNBC; it exploits a total of 1942 TNBC patient data collected from 19 datasets: 1243 samples from microarray technology (14 datasets) and 699 from RNAseq technology (five datasets including a new one from our institution [GSE225002]). We performed independent unsupervised analyses on these two large cohorts with consensus k-means clustering including selection of optimal number of clusters and optimal number of variables. Both consensus clustering analyses separated TNBC microarray and RNAseq TNBC cohorts into four optimal clusters. These clusters were then characterized using clinicopathological features, gene ontology enrichment analyses (GOEA), gene set enrichment analysis (GSEA), 70 gene expression signatures (GES) and expression of 47 immune checkpoint genes. We concluded that TNBC are composed of four robust subtypes: LAR, mesenchymal-like immune-activated (MLIA), BLIA and BLIS. Importantly, we propose that MLIA acronym should replace M or MSL, because it highlights the two main biological characteristics of this subtype, i.e. mesenchymal-like features and a high IR comparable to that of BLIA. Furthermore, we showed that each subtype shares common biological characteristics with another one: LAR and MLIA (androgen receptor pathway activation); MLIA and BLIA (immune activation, notably high levels of immune checkpoints); BLIA and BLIS (basal-like features); BLIS and LAR (immune suppression). As regards microarray cohort, LAR included 19.23% (n = 239) of TNBC, MLIA 17.38% (n = 216), BLIA 32.50% (n = 404) and BLIS 30.89% (n = 384). Concerning RNAseq cohort, LAR included 19.60% (n = 137) of TNBC, MLIA 17.31% (n = 121), BLIA 28.47% (n = 199) and BLIS 34.62% (n = 242). Distributions of patients among the four clusters in the two cohorts were similar (p = 0.3583). Distributions were also similar between studies for microarray cohort (p = 0.2479) and RNAseq cohort (p = 0.1775). Pooled data (n = 1942 patients) gave the following distribution: 19.36% for LAR; 17.35% for MLIA; 31.06% for BLIA; 32.23% for BLIS, confirming that TNBC are largely but not exclusively basal-like (63.29%). In brief, according to the TNBC subtype molecular characterisation, anti-androgen therapy could be prescribed for LAR, immune checkpoint inhibitors for MLIA and BLIA, alone or in combination, and TAM-focused and antineurogenic therapies for BLIS. As an extension of this work, TNBC transcriptomic data associated with their clinicopathological features and TNBC subtype annotations were included in bc-GenExMiner v5.0 web tool (http://bcgenex.ico.unicancer.fr). Citation Format: Mario Campone, Pascal Jézéquel, Fadoua Ben Azzouz, Wilfried Gouraud, Agnes Bassevile, Bertrand Michel, Hamza Lasla, Jean Sebastien FRENEL. Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-05.

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

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p&amp;lt; 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-07.

Clinical Advances in Triple Negative Breast Cancer Treatment: Focus on Poly (L-lactide-coglycolide) Nanoparticles.

Triple negative breast cancer (TNBC) is the most aggressive type of breast cancer and is associated with high probability of metastasis and poor prognosis. Chemotherapeutics and surgery remain the most common options for TNBC patients; however, chemotherapeutic resistance and relapse of tumors limit the progression free survival and patient life span. This review provides an overview of recent chemotherapeutics that are in clinical trial, and the combination of drugs that are being investigated to overcome the drug resistance and to improve patient survival in different molecular subtypes of TNBCs. Nanotherapeutics have emerged as a promising platform for TNBC treatment and aim to improve the selectivity and solubility of drugs, reduce systemic side effects, and overcome multi-drug resistance. The study explores the role of nanoparticles for TNBC treatment and summarizes the types of nanoparticles that are in clinical trials. Poly(L-lactide-co-glycolide) (PLGA) is the most studied polymeric carrier for drug delivery and for TNBC treatment in research and in clinics. This review is about providing recent advancements in PLGA nanotherapeutic formulations and their application to help treat TNBC. Some background on current chemotherapies and pathway inhibitors is provided so that the readers are aware of what is currently considered for TNBC. Some of the pathway inhibitors may also be of importance for nanotherapeutics development. SIGNIFICANCE STATEMENT: This minireview summarizes the progress on chemotherapeutics and nanoparticle delivery for treatment of TNBC and specifically highlights the lead compounds that are in clinical trials.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [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 6620.

A Pilot Study on BRCA1/2 and PI3K Mutations Across Subtypes of Triple Negative Breast Cancer in North Indian Population.

BRCA1/2 are tumor suppressor genes which regulate the DNA repair mechanism. Mutations in BRCA1/2 may increase the risk of breast cancer in patients. In the present study frequency of BRCA1/2 mutations in triple negative breast cancer (TNBC) patients was assessed and correlated with molecular subtypes of TNBC. Blood samples from 65 confirmed cases of TNBC were collected. DNA was isolated from whole blood and libraries were prepared using a BRCA1/2 custom panel. Sequencing was done on Ion torrent S5 sequencer and ion reporter was used for data analysis. Further molecular subtyping of mutation positive TNBC cases was done using immunohistochemistry markers CK5/6; CK4/14; Vimentin and E-Cadherin and androgen receptor (AR) using tissue microarray. Twenty five of 65 patients had heterozygous pathogenic mutations, alterations with conflicting interpretation of pathogenicity, variants of uncertain significance and variants of unknown significance. Nine patients had pathogenic mutation in BRCA 1 gene only and 2 patients had pathogenic mutations in BRCA2 gene. Two patients were transheterozygous for BRCA mutations, that is, had pathogenic mutations in both BRCA1/2 genes simultaneously and 5 were compound heterozygous (involving BRCA2 gene in all the cases). Prevalent subtypes among BRCA positive cases were unclassified subtype (n=4, 33%), Basal like (n=5, 41%), and mesenchymal subtype (n=3, 25%). None of the LAR subtype showed BRCA1/2 mutations. The present study observed that the BRCA1 mutation is more frequent than BRCA2 mutation in TNBC. BRCA1/2 mutations do not correspond to BRCAness or basal phenotype. Considering high incidence of breast cancer and lack of correlation of basal morphology with BRCA1/2 mutation, the molecular methods should be used for screening for BRCA1/2 mutations. This will not only help in familial screening but also in deciding targeted therapy with PARP (poly-ADP ribose polymerase) inhibitors.

176P Multiplex-immunoflourescence spatial patterns to predict triple-negative breast cancer molecular subtypes in the IMMUcan study

176P Multiplex-immunoflourescence spatial patterns to predict triple-negative breast cancer molecular subtypes in the IMMUcan study

Abstract B046: Analyzing heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 7 breast cancer scRNAseq studies

Abstract Breast cancer can be classified into several types. Among them, triple-negative breast cancer (TNBC) is breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Since targeted therapy for breast cancer relies on the three receptors, treatment outcomes have been worst in TNBCs. However, the clinical benefit of immune-checkpoint inhibitors (ICIs) with chemotherapy over chemotherapy alone was demonstrated in several trials, opening a new avenue for patients with metastatic PD-L1+ TNBC. After several studies with ICIs, researchers found that the tumor microenvironment affects the response rate of immunotherapies. Therefore, to unravel the heterogeneity of the tumor microenvironment of TNBC patients, we collected 7 public single-cell RNA sequencing breast cancer studies. From 141,068 cells populating tumors and tumor-microenvironment (TMEs), we first identified epithelial cells separately. Using whole cells including epithelial and immune-stromal cells, we identified the Baylor-proposed TNBC molecular subtype of each patient. This approach with epithelial cells revealed that 19 basal-like immune-activated (BLIA), 5 basal-like immune-suppressed (BLIS), 2 luminal-androgen receptor (LAR), 3 mesenchymal (MES), and 3 unclassified subtypes. By comparing cell number and cell-cell interactions, the heterogeneity of TME-consisting cell populations between the TNBC subtypes was analyzed. Analysis of tumor-infiltrating lymphocytes revealed their difference in activation, expansion, and exhaustion programs across patients. Among the subtypes, the BLIA subtype had a greater amount of exhausted CD8+ T cells and regulatory T cells. This finding was consistent with cell-cell interaction analysis. In the case of the BLIA subtype, the interaction between regulatory T cells and CD8 T cells was more active than in other subtypes. Also, the interaction between cytotoxic cells (T cells and NK cells) and regulatory T cells were more active in the BLIA subtype. Additionally, tumor cells in the BLIA subtype seem to inhibit cytotoxic cells to a greater extent. Among the interactions between epithelial cells and exhausted CD8+ T cells, MDK interaction was higher in the BLIA subtype. Additionally, the expression of the MDK gene in endothelial cells and macrophages leads to upregulation in immune infiltration which causes more T cells in TME. Also, we found that the progression-free survival of patients differs due to the expression of the MDK gene. In conclusion, the MDK gene causes a T cell-enriched environment in TME, which leads to different progression-free survival. Citation Format: Kyungsoo Kim, Jee Hung Kim, Soong June Bae, Sung Gwe Ahn, Joon Jeong. Analyzing heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 7 breast cancer scRNAseq studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B046.

Selective inhibition of CDK9 in triple negative breast cancer

Targeted therapy for triple-negative breast cancers (TNBC) remains a clinical challenge due to tumour heterogeneity. Since TNBC have key features of transcriptionally addicted cancers, targeting transcription via regulators such as cyclin-dependent kinase 9 (CDK9) has potential as a therapeutic strategy. Herein, we preclinically tested a new selective CDK9 inhibitor (CDDD11-8) in TNBC using cell line, patient-derived organoid, and patient-derived explant models. In vitro, CDDD11-8 dose-dependently inhibited proliferation (IC50 range: 281–734 nM), induced cell cycle arrest, and increased apoptosis of cell lines, which encompassed the three major molecular subtypes of TNBC. On target inhibition of CDK9 activity was demonstrated by reduced RNAPII phosphorylation at a CDK9 target peptide and down-regulation of the MYC and MCL1 oncogenes at the mRNA and protein levels in all cell line models. Drug induced RNAPII pausing was evident at gene promoters, with strongest pausing at MYC target genes. Growth of five distinct patient-derived organoid models was dose-dependently inhibited by CDDD11-8 (IC50 range: 272–771 nM), including three derived from MYC amplified, chemo-resistant TNBC metastatic lesions. Orally administered CDDD11-8 also inhibited growth of mammary intraductal TNBC xenograft tumours with no overt toxicity in vivo (mice) or ex vivo (human breast tissues). In conclusion, our studies indicate that CDK9 is a viable therapeutic target in TNBC and that CDDD11-8, a novel selective CDK9 inhibitor, has efficacy in TNBC without apparent toxicity to normal tissues.

Development of an ubiquitin-proteasome system signature for predicting prognosis and providing therapeutic guidance for patients with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a pathological subtype with a high mortality, and the development of inhibitors in the ubiquitin-proteasome system (UPS) component could be a novel therapeutic tool. Triple-negative breast cancer data were obtained from The Cancer Genome Atlas (TCGA), and subtype analysis was performed by consistent clustering analysis to identify molecular subtypes of TNBC according to UPS characteristics. Differential analysis, COX and least absolute shrinkage and selection operator (LASSO) COX regression analyses were performed to select genes associated with overall survival in TNBC. The final prognostic model (UPS score) was determined using the LASSO COX model. The model performance was assessed using receiver operating characteristic (ROC) curves and survival curves. In addition, the results of the UPS score on analyzing the abundance of immune cell infiltration and immunotherapy were explored. Finally, we developed a nomogram for TNBC survival prediction. Two UPS subtypes (UPSMS1 and UPSMS2) showing significant survival differences were classified. COX regression analysis on differentially expressed genes in UPSMS1 and UPSMS2 filtered five genes that affected overall survival. Based on the regression coefficients and expression data of the five genes, we built a prognostic assessment system (UPS score). The UPS score showed consistent prognostic and therapeutic guidance values. Finally, the ROC curve of the nomogram and UPS score showed the highest predictive efficacy compared with traditional clinical prognostic indicators. The UPS score represented a promising prognostic tool to predict overall survival and immune status and guide personalized treatment selection in TNBC patients, and this study may provide a more practical alternative for clinical monitoring and management of TNBC.

Research Progress on Molecular Subtyping and Modern Treatment of Triple-Negative Breast Cancer.

Breast cancer has become the most common malignant tumor worldwide. Triple-negative breast cancer (TNBC) is a type of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Compared with other molecular subtypes of breast cancer, TNBC is the most aggressive and highly heterogeneous. TNBC is insensitive to endocrine and anti-HER2 therapy, and chemotherapy is currently the main systemic treatment. With the continuous development of detection techniques and deepening research on TNBC molecular subtypes, drugs targeting immune checkpoints and different targets have emerged, such as atezolizumab, pembrolizumab, poly (ADP-ribose) polymerase (PARP) inhibitors, trophoblast cell-surface antigen 2 (TROP-2), and antibody-drug conjugates. These therapies provide new hope for TNBC treatment. Based on the analysis and classification of TNBC, this article summarizes the immunotherapy, targeted therapy, and new treatment combinations, providing references for the precise treatment of TNBC in the future.

Targeting DCLK1 attenuates tumor stemness and evokes antitumor immunity in triple-negative breast cancer by inhibiting IL-6/STAT3 signaling

Triple-negative breast cancer (TNBC) exhibits the poorest outcomes among breast cancer subtypes due to the high heterogeneity and a lasting scarcity of effectual treatments. Targeted therapies based on molecular subtypes of TNBC are critical step toward tailoring treatments to improve clinical outcomes. Gastrointestinal cancer stem cell (CSC) marker DCLK1 was reported to be highly expressed in stem cell-rich subtype of TNBC. Here, we firstly explored the impacts of DCLK1 on tumor cells as well as their immune microenvironment in TNBC and potential therapeutic strategies for TNBC patients with high DCLK1 expression. Our results disclosed that DCLK1 overexpression promoted, while knockout of DCLK1 suppressed the CSC-like traits of TNBC cells and resistance to chemotherapeutics. Besides, DCLK1 supported immune escape by inhibiting intratumoral cytotoxic T cell infiltration in TNBC and hence limited immune checkpoint inhibitors efficacy. Mechanistically, bioinformatics analysis revealed that IL-6/STAT3 signaling was significantly enriched in high DCLK1-expressing patients, and our results further revealed that DCLK1 enhanced IL-6 expression and STAT3 activation in TNBC cells, which finally gave rise to upregulated CSC traits and suppressed CD8+ T-cell activity. Inhibiting IL-6/STAT3 pathway by IL-6R antagonist, Tocilizumab or STAT3 inhibitor, S31-201 could abolish DCLK1-promoted malignant phenotypes of TNBC cells. Finally, DCLK1 was identified to be specifically and highly expressed in the mesenchymal-like subtype of TNBC and targeting DCLK1 could improve chemotherapy efficacy and activate antitumor immunity. Overall, our study revealed the potential clinical benefits of targeting DCLK1 in TNBC treatment.

Abstract P6-01-05: Platelet-derived growth factor-CC expression in primary triple-negative breast cancer is associated with the basal-like molecular subtype and increased immune infiltrate

Abstract Around 10-15% of all breast cancers are categorized clinically as triple-negative breast cancer (TNBC). TNBC is defined by lack of protein expression or over expression of treatment targets such as the Estrogen Receptor α (ER) and Human Epidermal Growth Factor Receptor-2 (HER2), and the prognostic factor PR (Progesterone Receptor). The negative definition of TNBC results in a heterogeneous mix of tumors with variable molecular characteristics and prognosis. Defining TNBC molecular subtypes in detail is of importance to improve prognostication and find new treatment options. Expression of Platelet-derived growth factor-CC (PDGF-CC) has previously been correlated with the TNBC subtype, and paracrine PDGF-CC signaling has been reported to be of importance for maintaining TNBC tumor cell phenotype. We aimed to characterize PDGF-CC expression within the TNBC patient population by combining studies of PDGF-CC in tissue microarrays (TMAs) with matching RNAseq data and clinical follow-up; all variables originating from the SCAN-B (Sweden Cancerome Analysis Network – Breast) clinical study (ClinicalTrials.gov: NCT02306096). TMAs constructed of primary TNBC patient samples were stained for PDGF-CC using the Dako PT Autostainer system. Tumor cell-specific expression of PDGF-CC intensity was scored as either absent (N=11), weak (N=86), intermediate (N=81) or strong (N=70), and the scores were used to create corresponding TNBC PDGF-CC subgroups. We then explored associations of these subgroups with clinicopathological variables and time-to-event outcomes. Intermediate and strong PDGF-CC scores were associated with Nottingham Histological Grade 3 (p=0.001), increased proliferation (p&amp;lt; 0.001) and younger patient age at diagnosis (p=0.002). RNAseq data corresponding to tumors included in the TMAs was then retrieved, and differentially expressed genes were identified and used to perform Gene Set Enrichment Analysis (GSEA) comparing the TMA-derived PDGF-CC subgroups. Immune-related signatures were found to be enriched in the strong PDGF-CC subgroup vs. intermediate. Interestingly, strong PDGF-CC intensity was associated with a decreased risk of recurrence in the chemotherapy treated patient group (HR 0.28, 95% CI 0.10-0.80, p=0.017). Finally, patient samples were assigned a PAM50 subtype and a TNBC molecular subtype by the TNBCtype algorithm. Ninety-four percent of tumors in the strong PDGF-CC subgroup were classified as basal-like, whereas the corresponding number in the weak and intermediate PDGF-CC subgroups were 51% and 84%, respectively. The TNBC molecular subtype termed ‘Immunomodulatory’ was more frequently represented in the strong PDGF-CC subgroup compared to weak and intermediate (33% vs. 13% and 16%, respectively). In conclusion, strong PDGF-CC protein expression identified basal-like TNBCs, with an increase in immune cell infiltrate shown by RNAseq analysis. Whether or not PDGF-CC has a direct effect on influx of immune cells into tumors remains to be investigated. Analyses are currently ongoing to better understand the improved outcome associated with strong PDGF-CC intensity and on the contrary, the worse outcome associated with weak and intermediate PDGF-CC intensity, and if paracrine PDGF-CC signaling may explain the discrepancy observed. Citation Format: Sophie Lehn, Gyula Pekar, Paulina Bolivar Balbas, Johan Staaf, Christina Möller, Kristina Lövgren, Anna Ehinger, Ana Bosch Campos, Åke Borg, Kristian Pietras. Platelet-derived growth factor-CC expression in primary triple-negative breast cancer is associated with the basal-like molecular subtype and increased immune infiltrate [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 P6-01-05.