Overall Survival In Triple-negative Breast Cancer Research Articles

Abstract PO4-24-01: Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and characterized by poor outcomes with a lack of targeted therapies. A comprehensive analysis of the molecular and immune landscape of invasive ductal TNBC can help improve our understanding of TNBC biology and identify novel targets and/or pathways for better management of this disease. Here, we characterized the molecular and immune signature of invasive ductal (ID) TNBC. Methods: 13,036 BC samples (ID TNBC, n=392; ID non-TNBC, n=927) were analyzed by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world overall survival (OS) and treatment-associated survival was extracted from insurance claims and calculated from tissue collection or treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: ID TNBC had a higher frequency of TP53, RB1, NF1, PIK3R1, NOTCH1, CREBBP, BRCA1, FANCI, and HRAS mutations (Table 1), NOTCH2 (4.3% vs 0.47%), EGFR (3.4% vs 0%), NFIB (4% vs 0.6%), MYB (3.5% vs 0.5%), CCNE1 (4.4% vs 1.1%) and AKT2 (2.4% vs 0.3%) copy number alterations, and NOTCH2 (1.6% VS 0.2%) fusion (all p < 0.05) compared to invasive ductal non-TNBC. Analysis of inferred immune cell infiltrates showed that ID TNBC had increased infiltration of Tregs (2% vs 1.7%), DC (3.2% vs 2.4%), CD8 T cells (0.5% vs 0.1%), and M1 macrophages (M) (3.4% vs 2.9%), but decreased infiltration of B cells (4.5% vs 6%) and M2M (2.9% vs 4.5%) (all p < 0.05). ID TNBC had increased T cell inflamed score (23 vs -9, p < 0.05), IFNy score (-0.24 vs -0.34, p < 0.05), MHC class I genes (HLA-A, HLA-B, TAP1, TAP2, FC: 1.1-1.5, all p < 0.05), immune checkpoint genes (CD274, PDCD1, CTLA4, PD-L2, FOXP3, IDO, FC: 1.1-2, all p < 0.05) and PD-L1 protein expression (SP142: 49.8% vs. 28.4%; 22c3: 41.7% vs. 16.8%, all p < 0.05). ID TNBC had differential expression of BCL2 family genes (upregulation: BAK1, BID, MCL1, BCL2A1, BCL2L10, FC: 1.1-2.0; downregulation: BAD, BIK, BBC3, BCL2, BCL2L1, BCL2L2, FC: 1.2-2.8, all p < 0.05) compared to invasive ductal non-TNBC. ID TNBC was associated with worse OS compared to ID non-TNBC (mOS: 24.5 vs 54.6 months; HR: 0.5, 95% CI 0.47-0.57; p < 0.00001) but trend towards better survival with pembrolizumab (mOS: 29.4 vs 8.8 month; HR: 0.67, 95% CI 0.28-1.5; p=0.3) treatment. No significant OS difference was noted for by high vs. low pro and anti-apoptotic BCL2 genes except for anti-apoptotic BCL2A1 (higher expression associated with better OS (mOS: 5.7 months, HR: 0.78, 95% CI 0.6-0.99; p=0.047). Conclusion: These data indicate that ID TNBC is associated with distinct mutational profile compared to non-TNBC, has increased T cell inflamed score, IFNy score, MHC class I and immune checkpoint gene expression, and differential immune cell infiltration. Interestingly, TNBC was associated with increased M1 and decreased M2M. There is evidence to suggest that transcriptomically defined M1 high tumors are clinically aggressive and have worse OS in TNBC. BCL2 family genes are not prognostic for TNBC outcomes except BCL2A1, which needs further prospective validation. Table 1: Mutation frequency in invasive ductal TNBC and invasive ductal non-TNBC Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Jose Leone, Priya Jayachandran, Stephanie Graff, Matthew Oberley, George Sledge Jr, Asher Chanan-Khan. Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-01.

SIRT3 Expression Predicts Overall Survival and Neoadjuvant Chemosensitivity in Triple-Negative Breast Cancer.

The Sirtuin (SIRT) family consists of seven evolutionary conserved NAD-dependent deacetylases that play important roles in various cancers, including breast cancer (BC). SIRTs expression has been reported to have prognostic value in BC, but these studies used limited sample size and yielded inconsistent conclusions. This study evaluated the association of SIRT3 and other SIRT family members with survival and neoadjuvant chemotherapy outcomes. BC patients' data was obtained from the TCGA-BRCA, METABRIC and GEO databases, comprising 4336 samples. SIRTs expression and overall survival (OS) were analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. SIRT3 expression levels were compared between pathologic complete response (pCR) and non-pCR groups after neoadjuvant chemotherapy in triple-negative breast cancer (TNBC). Protein-protein interaction networks were constructed using the STRING database. Gene set enrichment analysis (GSEA) was performed to explore potential functions of SIRT3. Through systematic analysis of SIRTs expression and OS of BC using three independent cohorts: TCGA-BRCA, METABRIC and GSE16446, we found that high SIRT3 expression was significantly associated with worse OS in TNBC in the TCGA-BRCA cohort, which was validated in the METABRIC and GSE16446 cohorts. SIRT3 expression was correlated with BC subtypes and American Joint Committee on Cancer (AJCC) T stage, but not with age-at-diagnosis, race, or tumor stage. Moreover, TNBC patients with higher SIRT3 expression had lower pCR rates after neoadjuvant chemotherapy (p = 6.40e-03) and SIRT3 expression was significantly lower in the pCR group than in the non-pCR group in TNBC (p = 4.2e-03). GSEA indicated that SIRT3 was involved in drug-related pathways such as oxidative phosphorylation, metabolism of xenobiotics by cytochrome P450, and drug metabolism. Our study suggests that SIRT3 is a potential biomarker for both OS and neoadjuvant chemosensitivity in TNBC. It may also assist in selecting suitable candidates and treatment options for TNBC patients.

Prognostic Values of CD8+, PARP, and EGFR on Overall Survival in Patients with Triple-Negative Breast Cancer.

This study aimed to investigate the associations of CD8+, PARP, and EGFR expressions with two-year survival in patients with triple-negative breast cancer (TNBC). A retrospective cohort study was conducted in a national cancer center. All patients aged 18 years diagnosed with TNBC (2013-2017) were included and followed for 24 months or until the patients were deceased. Kaplan-Meier survival function and Cox proportional hazard model were applied for the analyses. The study population was followed for 24 months (2,692 person-months, N = 126). At the end of the follow-up, 27 patients were deceased. The two-year mortality rate was 10 per 1,000 person-month. Kaplan-Meier graphs showed that after approximately one year of follow-up, poorer survival was seen in patients with low CD8+, positive PARP, and positive EGFR. The adjusted analysis found staging as the main predictor of overall survival in TNBC (HR = 7.20, 95% CI= 2.07 - 25.00). Patients with low CD8+, positive PARP, and positive EGFR expressions seem to be associated with poorer overall survival in TNBC. After approximately one year of follow-up, higher survival was observed in patients with high CD8+, negative PARP, and negative EGFR. Staging remains the main predictor of TNBC survival. Therefore, early detection and treatment of TNBC are essential to improve survival.

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.

Correlation analysis to assess surrogate endpoints for overall survival (OS) in triple-negative breast cancer (TNBC).

e13089 Background: The use of surrogate endpoints can help reduce the trial duration and cost which is important for aggressive diseases such as TNBC. Previous studies suggest progression-free survival (PFS) may serve as a surrogate for OS in metastatic TNBC. We aim to evaluate PFS and additional surrogate endpoints for their correlation with OS in localized and advanced TNBC. Methods: TNBC phase 3 trials and pooled analysis as well as trials including TNBC subgroups, were identified through a systematic search using LARVOL CLIN, PubMed, clinicaltrials.gov, and conference proceedings. Out of these, trials reporting median survival and hazard ratios (HR) for both OS and surrogate endpoints were considered for further statistical analysis. Surrogate endpoints reported by fewer than 4 trials were excluded from the study. Pearson correlation (r) and determination coefficient (R2) were calculated to assess surrogacy between Median OS and Median PFS, HR OS and HR PFS, HR disease-free survival (DFS), and invasive disease-free survival (IDFS). Weighted regression analysis was conducted controlling for sample size among trial arms. Results: 26 TNBC cohorts and 2 pooled analyses evaluating PARP, PD-1, PD-L1 inhibitors, and chemotherapies were included. The association analysis results are shown. There was a significant correlation between median OS and median PFS (r = 0.73, p value = 1.026×10−6) as well as between HR OS and HR PFS (r = 0.61, p value = 0.009). There was also a significant correlation between HR DFS and HR IDFS with HR OS (r = 0.92, p value = 0.003; r = 0.99, p value = 0.014). Conclusions: PFS is significantly correlated with OS in TNBC across diverse therapies. Also, HR PFS, HR DFS, and HR IDFS are significantly correlated with HR OS. Overall, these results illustrate that PFS could be used as a surrogate for OS in TNBC. Subsequent studies should include early-phase trials to increase association robustness and enable correlation comparison between the diverse mechanism of actions and other surrogate endpoints. [Table: see text]

Immune checkpoints expression patterns in early-stage triple-negative breast cancer predict prognosis and remodel the tumor immune microenvironment.

Currently, targeting immune checkpoint molecules holds great promise for triple-negative breast cancer (TNBC). However, the expression landscape of immune checkpoint genes (ICGs) in TNBC remains largely unknown. Herein, we systematically investigated the ICGs expression patterns in 422 TNBC samples. We evaluated the ICGs molecular typing based on the ICGs expression profile and explored the associations between ICGs molecular subtypes and tumor immune characteristics, clinical significance, and response to immune checkpoint inhibitors (ICIs). Two ICGs clusters and two ICGs-related gene clusters were determined, which were involved in different survival outcomes, biological roles and infiltration levels of immune cells. We established a quantification system ICGs riskscore (named IRS) to assess the ICGs expression patterns for individuals. TNBC patients with lower IRS were characterized by increased immune cell infiltration, favorable clinical outcomes and high sensitivity to ICIs therapy. We also developed a nomogram model combining clinicopathological variables to predict overall survival in TNBC. Genomic feature analysis revealed that high IRS group presented an increased tumor mutation burden compared with the low IRS group. Collectively, dissecting the ICGs expression patterns not only provides a new insight into TNBC subtypes but also deepens the understanding of ICGs in the tumor immune microenvironment.

The Role of Apoptotic Genes and Protein-Protein Interactions in Triple-negative Breast Cancer.

Compared to other breast cancer types, triple-negative breast cancer (TNBC) has historically had few treatment alternatives. Therefore, exploring and pinpointing potentially implicated genes could be used for treating and managing TNBC. By doing this, we will provide essential data to comprehend how the genes are involved in the apoptotic pathways of the cancer cells to identify potential therapeutic targets. Analysis of a single genetic alteration may not reveal the pathogenicity driving TNBC due to the high genomic complexity and heterogeneity of TNBC. Therefore, searching through a large variety of gene interactions enabled the identification of molecular therapeutic genes. This study used integrated bioinformatics methods such as UALCAN, TNM plotter, PANTHER, GO-KEEG and PPIs to assess the gene expression, protein-protein interaction (PPI), and transcription factor interaction of apoptosis-regulated genes. Compared to normal breast tissue, gene expressions of BNIP3, TNFRSF10B, MCL1, and CASP4 were downregulated in UALCAN. At the same time, BIK, AKT1, BAD, FADD, DIABLO, and CASP9 was down-regulated in bc-GeneExMiner v4.5 mRNA expression (BCGM) databases. Based on GO term enrichment analysis, the cellular process (GO:0009987), which has about 21 apoptosis-regulated genes, is the top category in the biological processes (BP), followed by biological regulation (GO:0065007). We identified 29 differentially regulated pathways, including the p53 pathway, angiogenesis, apoptosis signaling pathway, and the Alzheimer's disease presenilin pathway. We examined the PPIs between the genes that regulate apoptosis; CASP3 and CASP9 interact with FADD, MCL1, TNF, TNFRSRF10A, and TNFRSF10; additionally, CASP3 significantly forms PPIs with CASP9, DFFA, and TP53, and CASP9 with DIABLO. In the top 10 transcription factors, the androgen receptor (AR) interacts with five apoptosis-regulated genes (p<0.0001; q<0.01), followed by retinoic acid receptor alpha (RARA) (p<0.0001; q<0.01) and ring finger protein (RNF2) (p<0.0001; q<0.01). Overall, the gene expression profile, PPIs, and the apoptosis-TF interaction findings suggest that the 27 apoptosis-regulated genes might be used as promising targets in treating and managing TNBC. Furthermore, from a total of 27 key genes, CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were significantly correlated with poor overall survival in TNBC (p-value <0.05); they could play important roles in the progression of TNBC and provide attractive therapeutic targets that may offer new candidate molecules for targeted therapy. Our findings demonstrate that CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were substantially associated with the overall survival rate (OS) difference of TNBC patients out of a total of 27 specific genes used in this study, which may play crucial roles in the development of TNBC and offer promising therapeutic interventions.

Real-world evidence on predictors of survival for hormone-positive and triple-negative advanced breast cancer by treatment and BRCA status in the United States.

e18754 Background: Factors that affect overall survival (OS) for women with metastatic or recurrent breast cancer (BC) and BRCA1 or BRCA2 mutations (BRCA+) are a matter of debate. We analyzed electronic health records (EHR) from centers across the US to examine the effect of bilateral salpingo oophorectomy (BSO) and other factors on OS across treatment and molecular subtypes with emphasis on hormone-positive (HR+) and triple-negative BC (TNBC). Methods: A cohort of women diagnosed with stage 3 or 4 HR+ or TNBC from 1-Jan-2015 to 31-Dec-2019 was identified from EHR (Tempus Labs, Chicago, IL). BRCA+ was confirmed from medical notes and laboratory reports. A comparator group included HR+ and TNBC with no record of BRCA testing (Non-BRCA). Kaplan-Meier and Cox proportional hazards were used to evaluate predictors of OS from earliest diagnosis in multivariate models by BRCA status and molecular phenotype; treatment exposure (any time), mastectomy and BSO were treated as time-dependent variables. Results: We identified 1227 women with median age of 53 years (range 18-89) at BC diagnosis and median follow up of 26.1 months (max &gt; 26 years) and 358 deaths. Two-thirds had distant visceral or bone metastases, almost 60% were recurrent after original stage 1-3 BC; 302 women were stage 3 and 296 stage 4 at BC. The BRCA+ cohort included 439 women (195 BRCA1, 220 BRCA2, 24 “BRCA” mutation; 265 HR+, 142 TNBC) and median age at BC was 46 years (44 BRCA1, 47 BRCA2). Non-BRCA cohort consisted of 788 women with a median age of 57 at BC (378 HR+, 341 TNBC). BRCA+ had longer OS than non-BRCA ( P= 0.0001); HR+ had longer OS than TNBC ( P= 0.0001). For 331 TNBC, chemotherapeutic regimens were most common first line treatment; few (17%) received third line and 31% had no evidence of receiving systemic therapy. Age ≤50, BRCA+, and mastectomy were associated with increased OS in HR+; BRCA+ status and mastectomy were predictors of increased OS whereas platinum-based chemotherapy was associated with decreased OS in TNBC (Table). Across molecular subtypes, BSO was not an independent predictor of OS and most women (92%) had BSO after BC diagnosis. Conclusions: Regardless of BRCA status, women with advanced TNBC were almost exclusively treated with chemotherapy, which highlights the remaining unmet need for effective treatment options. Taking timing of BSO and other interventions into account is critical to understanding their effects of BC mortality.[Table: see text]

Expression of Proteinase-activated Receptor 2 (PAR2) as a Correlate of Concern in Triple-negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC), a highly aggressive cancer with poor outcome and lacking specific diagnostic, prognostic, or targeted therapeutic strategies, constitutes roughly 20% of all breast cancer cases. TNBC cells lack receptors for estrogen, progesterone, and human epidermal growth factor. The effort continues to find a suitable correlate that could serve as a TNBC biomarker, or as therapeutic target, or both. A retrospective study was performed with 88 TNBC and 74 non-TNBC patients who had undergone mastectomy/lumpectomy with axillary clearance for carcinoma breast. Immunohistochemical staining was carried out for levels of proteinase-activated receptor 2 (PAR2), encoded by F2RL1 gene, and staining scores were calculated, based on intensity and percentage positivity. PAR2 levels were markedly upregulated in TNBC patients, compared with patients with other breast cancer subtypes. Amongst different non-TNBC subtypes, higher expression was noted in luminal B (88.8%) and HER2+ (100%), compared with luminal A (52.5%). PAR2 levels were significantly high in TNBC patients with age more than 40 years than corresponding patients of non-TNBC group (P=0.0017). Furthermore, there was a statistically significant increase in levels of PAR2 expression in lymph node negative (P=0.0096) and early stage (P=0.005) of TNBC versus non-TNBC patients. PAR2 staining of ductal carcinoma in situ and invasive ductal carcinoma revealed lower expression in invasive component. Our data suggest that PAR2 levels constitute a correlate of concern for TNBC, tying in with a recent report that higher levels of F2RL1 gene expression correlate with poorer disease-free, as well as overall survival in TNBCs.

Abstract P4-07-21: High Tinagl1 expression is a marker of good prognosis in breast cancer

Abstract Background: Tubulointerstitial nephritis antigen-like 1 (Tinagl1) is reported to inhibit integrin and epidermal growth factor receptor (EGFR) and suppress tumor metastasis and growth in breast cancer. We investigated the association between Tinagl1 mRNA expression and clinicopathological factors and prognosis of breast cancer patients with long-term follow-up. The median follow-up period was 12.0 years. Materials and Methods: A total of 599 consecutive primary invasive breast cancer patients with available tissue from surgery in our hospital from 1992 to 2008 were included in this study. We examined the association between Tinagl1 mRNA expression and prognostic and clinicopathological factors using the TaqMan real-time PCR system. Statistical analysis was performed using the Kaplan-Meier method and the Cox proportional hazards model. Results: High expression of Tinagl1 mRNA was associated with significantly better disease-free survival (DFS) and overall survival (OS) than low expression (P=0.003 and P=0.01, respectively). In estrogen receptor (ER)-positive patients, DFS and OS were also better in the high Tinagl1 mRNA expression group (P=0.01 and P=0.03, respectively). However, there was no significant difference in DFS and OS in triple-negative breast cancer. Low Tinagl1 mRNA expression was positively associated with higher tumor grade. Multivariate analysis showed that low Tinagl1 mRNA expression, together with lymph node positivity, was an independent poor prognostic factor in DFS (HR 1.48; 95% CI 1.06-2.05, P=0.02). Conclusions: Our findings suggest that Tinagl1 has a role in the metastasis of breast cancer, and that its expression could be a promising candidate biomarker of prognosis in breast cancer patients. Variablesn(%)DSF Univariate P valueDFS Multivariate P valueHR (95% CI)Nodal status590Negative349 (59.2)1Positive241 (40.8)&amp;lt;0.0001&amp;lt;0.00013.16(2.25-4.45)Tinagl1 mRNA expression599High300 (50.1)1Low299 (49.9)0.0030.021.48(1.06-2.05) Citation Format: Akiko Kato, Naoto Kondo, Yumi Wanifuchi-Endo, Takashi Fujita, Tomoko Asano, Tomoka Hisada, Yasuaki Uemoto, Mitsuo Terada, Tatsuya Toyama. High Tinagl1 expression is a marker of good prognosis in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-21.

Screening of DNA Damage Repair Genes Involved in the Prognosis of Triple-Negative Breast Cancer Patients Based on Bioinformatics.

Background: Triple-negative breast cancer (TNBC) is a special subtype of breast cancer with poor prognosis. DNA damage response (DDR) is one of the hallmarks of this cancer. However, the association of DDR genes with the prognosis of TNBC is still unclear.Methods: We identified differentially expressed genes (DEGs) between normal and TNBC samples from The Cancer Genome Atlas (TCGA). DDR genes were obtained from the Molecular Signatures Database through six DDR gene sets. After the expression of six differential genes were verified by quantitative real-time polymerase chain reaction (qRT-PCR), we then overlapped the DEGs with DDR genes. Based on univariate and LASSO Cox regression analyses, a prognostic model was constructed to predict overall survival (OS). Kaplan–Meier analysis and receiver operating characteristic curve were used to assess the performance of the prognostic model. Cox regression analysis was applied to identify independent prognostic factors in TNBC. The Human Protein Atlas was used to study the immunohistochemical data of six DEGs. The prognostic model was validated using an independent dataset. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis were performed by using gene set enrichment analysis (GSEA). Single-sample gene set enrichment analysis was employed to estimate immune cells related to this prognostic model. Finally, we constructed a transcriptional factor (TF) network and a competing endogenous RNA regulatory network.Results: Twenty-three differentially expressed DDR genes were detected between TNBC and normal samples. The six-gene prognostic model we developed was shown to be related to OS in TNBC using univariate and LASSO Cox regression analyses. All the six DEGs were identified as significantly up-regulated in the tumor samples compared to the normal samples in qRT-PCR. The GSEA analysis indicated that the genes in the high-risk group were mainly correlated with leukocyte migration, cytokine interaction, oxidative phosphorylation, autoimmune diseases, and coagulation cascade. The mutation data revealed the mutated genes were different. The gene-TF regulatory network showed that Replication Factor C subunit 4 occupied the dominant position.Conclusion: We identified six gene markers related to DDR, which can predict prognosis and serve as an independent biomarker for TNBC patients.

GCH1 induces immunosuppression through metabolic reprogramming and IDO1 upregulation in triple-negative breast cancer

PurposeRegulatory T cells (Tregs) heavily infiltrate triple-negative breast cancer (TNBC), and their accumulation is affected by the metabolic reprogramming in cancer cells. In the present study, we sought to identify...

Abstract 483: TIGIT/PVR/NECTIN immune checkpoint axis expression across breast cancer subtypes

Abstract Background: Targeting the PD1:PD-L1 pathway revolutionized treatment options for patients with metastatic triple negative breast cancer (TNBC). Other immune checkpoint inhibitors are being investigated in clinical settings, such as anti-TIGIT antibodies. The goal of this analysis was to evaluate the expression of the TIGIT/PVR/NECTIN molecule family in breast cancer subtypes. Methods: We used the publicly available METABRIC dataset (Curtis C, Nature 2012; Pereira B, Nature Comm 2016) to evaluate microarray RNA expression levels for TIGIT, CD226, PVR, PVRIG, NECTIN2, NECTIN3, NECTIN4, PDCD1 and CD274 from primary breast tumors annotated with ER, PR and HER2 IHC status (Hormone receptor positive [HR+]: n=1369; HER2 amplified [HER2+]: n=236; TNBC: n=299). Biomarker association with overall survival (OS) was evaluated using Cox proportional hazard analysis adjusted for clinical covariates. Results: Expression of the TIGIT/PVR/NECTIN members revealed three expression patterns across breast cancer subtypes: 1) highest in TNBC, followed by HER2+ and then by HR+ BC (PVR); 2) similar levels in TNBC and HER2+, and higher than HR+ BC (TIGIT, CD226, PVRIG, NECTIN4); 3) highest in HR+, followed by HER2+ and then by TNBC (NECTIN2). NECTIN3 showed the lowest expression differences across subtypes. Correlation of these genes with each other varied: 1) Receptors TIGIT, CD226 and PVRIG were moderately correlated with each other and with PDCD1 (Pearson correlation index [r]: 0.45 - 0.78) and strongly correlated with the T cell markers CD3E, CD8A, and GZMB (r: 0.53 - 0.90); 2) PVR family ligands PVR, NECTIN2, NECTIN3 and NECTIN4 were not correlated with each other, nor to the receptors or biomarkers for T-cell, macrophage or stromal biology (r: -0.36 - 0.30). CD274, the PD1 ligand, was weakly associated with TIGIT, CD226, PVRIG, PDCD1 and with immune cell markers (0.25 - 0.38). This pattern was similar across breast cancer subtypes. No OS prognostic value was observed for the TIGIT/PVR family members in HR+ or HER2+ BC. Only CD226 was associated with an improved OS in TNBC (HR: 0.69 [0.48-0.99]). As expected, PDCD1, CD274, and T-cell biomarkers were linked to improved OS prognosis (HR: 0.61-0.69), observed only in TNBC. Conclusions: Overall, RNA expression levels of TIGIT/PVR/NECTIN family members are higher in HER2+ and TNBC compared to HR+ in the large breast cancer METABRIC dataset. While TIGIT, CD226 and PVRIG are clearly associated with T cells, the cell subsets expressing the PVR/NECTIN ligands remain to be defined in breast cancer. Our analysis suggests that TNBC and HER2+ are the breast cancer subtypes more likely to benefit from anti-TIGIT therapeutic targeting. Citation Format: Kelly J. DuPree, Luciana Molinero. TIGIT/PVR/NECTIN immune checkpoint axis expression across breast cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 483.

Abstract P1-10-20: Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC)

Abstract Introduction High levels of TIL at baseline are associated with higher pCR rates and better overall survival in TNBC. Recent studies have also indicated that higher TIL in post-NACT residual disease in TNBC are an important independent predictor of improved survival. We evaluated the role of mid-treatment (post-AC; Adriamycin/Cyclophosphamide) TIL in predicting pCR rates in early-stage TNBC. Methods Of 242 patients with stage I-III TNBC enrolled in the ARTEMIS trial (NCT02276443), 156 patients had pre-AC TIL and pCR status available for this analysis. Both pre-and post-AC TIL counts were available in 29 patients. Post-AC TIL counts for the remaining patients were imputed using linear regression with age, race, stage III, vimentin &amp;gt;50% and post-AC tumor volume reduction. Using these imputed TIL counts we evaluated the association of post-AC TIL with pCR. We also evaluated the change in TIL before and after treatment with AC. Results At baseline the median TIL count was 10% (n=156). In the post-AC samples, the median TIL count was 5%. Using imputed TIL counts, we did not conclude that post-AC TIL was associated with pCR (p= 0.28). Using a cut-point of 15% TIL, our analysis showed that baseline TIL is more strongly correlated with pCR than post-AC TIL (Table 1). In our univariable logistic regression, both baseline TIL and the difference in TIL pre-and post- treatment were significantly associated with pCR (p= 0.0015 and p=0.0068, respectively), however in the multivariable analysis only baseline TIL was significant. Our analysis did show that a decrease in TIL from pre- to post-treatment was significantly associated with pCR (p=0.022). However, this measure was not significant in our logistic regression model when pre-TIL was also included. Conclusion Higher pre-treatment TIL correlated more strongly with pCR rate when compared to post-AC TIL. Pre-treatment high TIL was associated with pCR regardless of changes in TIL pre and post treatment. Table 1. Changes in TIL before and after treatmentBaseline TILPost-AC TILN#pCR (%)LowLow6217 (27%)LowHigh4012 (30%)HighLow2513 (52%)HighHigh2916 (55%)TIL; Low: &amp;lt;15, High &amp;gt;15 Citation Format: Nour Abuhadra, Kenneth Hess, Jennifer Litton, Gaiane Rauch, Alastair Thompson, Bora Lim, Beatriz Adrada, Elizabeth Mittendorf, Senthil Damodaran, Rosalind Candelaria, Banu Arun, Wei Tse Yang, Naoto Ueno, Lumarie Santiago, Rashmi Murthy, Nuhad Ibrahim, Sahin Aysegul, William Symmans, Lei Huo, Stacy Moulder. Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-20.

Gene and lncRNA co-expression network analysis reveals novel ceRNA network for triple-negative breast cancer

Breast cancer is the most frequently diagnosed malignancy among women, and triple-negative breast cancer (TNBC) is a highly aggressive subtype. Increasing evidence has shown that lncRNAs are involved in tumor growth, cell-cycle, and apoptosis through interactions with miRNAs or mRNAs. However, there is still limited data on ceRNAs involved in the molecular mechanisms underlying TNBC. In this study, we applied the weighted gene co-expression network analysis to the existing microarray mRNA and lncRNA expression data obtained from the breast tissues of TNBC patients to find the hub genes and lncRNAs involved in TNBC. Functional enrichment was performed on the module that correlated with Ki-67 status the most (Turquoise module). The hub genes in the Turquoise module were found to be associated with DNA repair, cell proliferation, and the p53 signaling pathway. We performed co-expression analysis of the protein-coding and lncRNA hub genes in the Turquoise module. Analysis of the RNA-seq data obtained from The Cancer Genome Atlas database revealed that the protein-coding genes and lncRNAs that were co-expressed were also differentially expressed in the TNBC tissues compared with the normal mammary tissues. On the basis of establishing the ceRNA network, two mRNAs (RAD51AP1 and TYMS) were found to be correlated with overall survival in TNBC. These results suggest that TNBC-specific mRNA and lncRNAs may participate in a complex ceRNA network, which represents a potential therapeutic target for the treatment of TNBC.

MiR-3178 inhibits cell proliferation and metastasis by targeting Notch1 in triple-negative breast cancer

Triple-negative breast cancer (TNBC) has a poorer outcome than other subtypes of breast cancer, and the discovery of dysregulated microRNA (miRNA) and their role in tumor progression has provided a new avenue for elucidating the mechanism involved in TNBC. In this study, we identified that miR-3178 was significantly reduced in TNBC, and the low miR-3178 expression correlated with poor overall survival in TNBC but not in non-TNBC. The ectopic overexpression of miR-3178 suppressed TNBC cell proliferation, invasion, and migration by inhibiting the epithelial-to-mesenchymal (EMT) transition. Notch1 was validated as the direct target gene of miR-3178, which was confirmed by the dual-luciferase reporter assay. miR-3178 decreased the expression of Notch1 and restoration of Notch1 expression attenuated the inhibitory effects of miR-3178 on cell proliferation, metastasis, and the EMT in TNBC. miR-3178 inhibited cell proliferation and metastasis by targeting Notch1 in TNBC, and the restoration of miR-3178 might be a potential therapeutic strategy for TNBC.

Tumor-infiltrating lymphocyte volume is a better predictor of neoadjuvant therapy response and overall survival in triple-negative invasive breast cancer

Triple-negative breast cancer (TNBC) has an aggressive behavior, limited therapeutic options, and high mortality rate. Neoadjuvant chemotherapy is a standard treatment for TNBC, and patients with pathological complete response (pCR) have a favorable outcome. We conducted a comprehensive evaluation of cancer clinicopathological parameters and correlated these parameters with the pCR rate and the overall survival. Fifty-eight patients with TNBC of no special type who underwent breast biopsy, neoadjuvant therapy, and mastectomy in our institution during 2005-2016 were included in this study. Among the 58 TNBC patients, 26 (45%) achieved pCR, and 32 (55%) had residual disease. The study parameters included age, histologic grade, clinical stage, mitotic count, Ki-67 proliferation index, stromal ratio, stromal type, tumor necrosis, stromal tumor-infiltrating lymphocytes (TILs), tumor intraepithelial lymphocytes, and tumor-infiltrating lymphocytes volume (TILV). Whereas most factors did not affect pCR, stromal TILs and TILV showed significant correlation with pCR (P = .01 and P = .0008, respectively). In the residual disease group, all factors showed no significant differences before and after neoadjuvant chemotherapy, except for tumor sizes. Lastly, pCR, TILs, and TILV were all significantly correlated with the overall survival, with P = .028, .029, and .015, respectively. In summary, we proposed a new concept of TILV to precisely evaluate the tumor immunity, and our data showed that TILV had a better predictive value than TILs for the pCR and the overall survival in TNBC.

PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer.

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

Abstract P3-07-09: ERRβ copy number and expression in triple negative breast cancer

Abstract Introduction: Triple negative breast cancer (TNBC) is a highly aggressive form of breast cancer prevalent in African-American (AA) women defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative. Because ER- and HER2-targeted therapies are ineffective in TNBC, systemic chemotherapy is the standard of care and there is a tremendous need for new effective therapies with less toxicity. Steroid hormone receptors are highly druggable targets, and orphan nuclear receptors, members of the nuclear receptor superfamily, are emerging as targets for cancer therapy. In fact, we have previously shown that treatment of TNBC cells with a small molecule agonist ligand (DY131) for estrogen related receptor beta (ERRβ), has growth inhibitory and anti-mitotic activity. We have also shown that increased mRNA expression of ERRβ, correlates with better recurrence- and distant metastasis-free survival in TNBC/basal-like breast cancer. The goal of our current work is to comprehensively characterize ERRβ copy number and mRNA status in TNBC and determine its association with patients' prognosis. Methods: ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by array-CGH, using the Agilent SurePrint G3 Human CGH platform. ESRRB mRNA data and its association with overall survival was determined in systemically untreated patients from METABRIC using Illumina gene expression array data (probe ID ILMN_1707398). Results: Copy number alterations (CNAs). Copy number losses at the ESRRB locus (14q24.3) were observed in 10/56 (17.8%) of TNBC vs. 10/50 (20%) of nonTNBC, while copy number gains were detected in 43/56 (76.8%) of TNBC vs. 29/50 (58%) of nonTNBC (c2 *p=0.036). Interestingly, in both TNBC and non-TNBC, ESRRB loss was seen with markedly higher frequency in AA patients when compared to Caucasian (CA) patients (c2 *p=0.012 for TNBC, p=0.052 for non-TNBC). mRNA expression. Among patients not treated with systemic chemotherapy in the METABRIC dataset, low ESRRB mRNA was significantly associated with shorter overall survival in TNBC, but not ER+ or HER2+ patients (TNBC hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Low ESRRB also correlated with reduced overall survival in TP53 mutant (but not wild type) tumors (hazard ratio 0.28, 95% confidence interval 0.1-0.82, *p=0.013). Conclusions: ESRRB presents significantly high levels of copy number losses in TNBC when compared to non-TNBC tumors. In breast tumors from AA women, both the TNBC and non-TNBC subtypes are significantly more likely to have reduced ESRRB copy number vs. CA women. Low ESRRB mRNA expression predicts for poor overall survival in TNBC and TP53 mutant tumors. These data advocate that ERRβ expression has prognostic value in breast cancer, particularly TNBC. Future goals include immunohistochemistry staining, and analysis, of a tissue microarray consisting of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+); as well as ERRβ overexpression and knock-down studies in TNBC cell lines to define the role it plays in TNBC. Citation Format: Fernandez AI, Graham G, Győrffy B, Cavalli L, Mahajan A, Riggins RB. ERRβ copy number and expression in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-09.

Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy

Tumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal-HER2-negative breast cancer. Patients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0-10% immune cells in stromal tissue within the tumour), intermediate (11-59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts. In the luminal-HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p<0·0001 for each subtype, χ2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87-0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89-0·99], p=0·017), but not in luminal-HER2-negative tumours (1·02 [0·96-1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86-0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86-1·02], p=0·11), and was associated with shorter overall survival in luminal-HER2-negative tumours (1·10 [1·02-1·19], p=0·011). Increased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal-HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted. Deutsche Krebshilfe and European Commission.