Negative Breast Cancer Subtype Research Articles

Mechanisms of S-phase arrest and mitochondrial dysfunction in complex III by DHODH inhibitors in tumorigenic TNBC cells.

Dihydroorotate dehydrogenase (DHODH) inhibitors have recently gained increasing research interest owing to their potential for treating breast cancers. We explored their effects in different breast cancer subtypes, focusing on mitochondrial dysfunction. The sensitivity of different subtypes to the inhibitors was investigated with respect to DHODH expression, tumorigenic, and receptor status. Analysis of respiratory complexes, cell cycle, reactive oxygen species (ROS), and cell differentiation were performed. Four cell lines with different receptor status were included, namely MCF-7, MDAMB-231, SKBR-3, and MCF-10A. We showed that MCF-7 and MDAMB-231 cells of the subtypes (ER+/PR+/HER2-) and (ER-/PR-/HER2-), respectively, were responsive to brequinar. Brequinar (BQR) caused cell cycle arrest in the S-phase in sensitive subtypes of breast cells but induced cell differentiation only in poorly differentiated breast cells. All cell subtypes showed increased generation of ROS, both intracellular and mitochondrial ROS with a greater increase seen in mitochondrial ROS in response to DHODH inhibitor, subsequently contributing to mitochondrial dysfunction. BQR also disrupts the function of complex III in ER+/PR+ and triple negative breast cancer (TNBC) subtypes. Collectively, we have found that MDAMB-231 TNBC cell was the most affected by DHODH inhibition in terms of sensitivity, cell cycle arrest, induction of cell differentiation, production of ROS, and mitochondrial complexes disruption. In conclusion, these findings suggest that DHODH inhibitors can potentially become a valuable targeted therapy for TNBC subtype and further consolidates its therapeutic potential as part of the combinatorial therapy against this resilient breast cancer subtype.

Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

TYRO3 and EPHA2 Expression Are Dysregulated in Breast Cancer.

Receptor tyrosine kinases (RTKs) are involved in cell growth, motility, and differentiation. Deregulation of RTKs signaling is associated with tumor development and therapy resistance. Potential RTKs like TAM (TYRO3, AXL, MERTK), RON, EPH, and MET have been evaluated in many cancers like lung, prostate, and colorectal, but little is known in breast tumors. In this study, 51 luminal breast cancer tissue and 8 triple negative breast cancer (TNBC) subtypes were evaluated by qPCR for the expression of TAM, RON, EPHA2, and MET genes. Statistical analysis was performed to determine the correlation to clinical data. TYRO3 is related to tumor subtype and stage, patient's age, smoking habits, and obesity. MET expression is correlated to EPHA2 and TAM gene expression. EPHA2 expression is also related to aging and smoking habits. The expression levels of the TAM and EPHA2 genes seem to play an important role in breast cancer, being also influenced by the patient's lifestyle.

NUAKs promote mTOR/c-Myc-induced glucose and glutamine reprogramming for cell growth and metastasis in breast cancer cells

Breast cancer progression and metastasis are closely connected to changes in glucose and glutamine metabolism. While Novel (nua) kinase family 1 (NUAK1) and Novel (nua) kinase family 2 (NUAK2), which are two members of the AMPK-related kinases, have been associated with breast tumorigenesis, their role in the metabolic reprogramming that occurs during breast cancer progression remains unclear. Our research uncovers that NUAKs expression is significantly higher in breast cancer tissues and cell lines, and it is positively related to glycolysis, the pentose phosphate pathway (PPP), glutamine metabolism, and a poor prognosis for breast cancer patients. We show that NUAKs significantly increase metabolic reprogramming, including aerobic glycolysis, PPP, and glutamine metabolism in triple negative breast cancer subtypes but only induce aerobic glycolysis and PPP in luminal breast cancer subtypes to meet the anabolic demands of rapidly dividing breast cancer cells. In contrast, the depletion of NUAKs has the opposite effect. Mechanistic insights reveal that NUAKs activate mammalian target of rapamycin (mTOR) signaling, which in turn upregulates the c-Myc transcription factor, a crucial regulator of glucose and glutamine metabolic gene expression. Moreover, we demonstrate that NUAKs enhance mTOR/c-Myc signaling pathways, leading to increased glucose and glutamine reprogramming, which supports rapid cell proliferation and metastatic potential in breast cancer cells. Importantly, pretreating breast cancer cells with mTOR inhibitors blocked the metabolic reprogramming and tumor-promoting effect of NUAK1/2. Therefore, targeting NUAKs may represent a novel therapeutic strategy for the treatment of breast cancer.

Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

Bioinformatic Investigation of Genetic Changes in Paraoxonase Genes in Breast Cancer and Breast Cancer Subtypes.

Among women, breast cancer (BC) is the most prevalent form of cancer. Many molecular targets have been discovered for BC prognosis and treatment. However, new markers still need to be identified, as cancer pathogenesis is triggered by different mechanisms. The aim of this study was to examine the changes in the paraoxonase genes (PON1, PON2, and PON 3) involved in the pathogenesis of BC. The characteristics of the mutations were evaluated with the Cbioportal database. Kaplan-Meier Plot evaluated recurrence-free survival (RFS). The UALCAN database determined the promoter methylation. Gene expression was evaluated by GEPIA2.0 database. PON1 harbored the most mutations. There was a significant decrease in PON3 expression levels in BC samples compared with healthy samples. PON1 and PON2 expression levels did not differ between BC tissue and normal adjacent tissue. Elevated expression levels of PON1 and PON2 genes were correlated with longer RFS, whereas reduced expression of the PON2 gene showed an association with longer RFS. Moreover, the promoter regions of PON1 and PON 3 were found to be hypermethylated, while the promoter region of PON 2 was found to be hypomethylated. The PON3 promoter region was significantly hypermethylated in luminal and human epidermal growth factor receptor 2 (HER2) + BC subtypes. However, the PON3 promoter region was significantly hypomethylated in the triple negative breast cancer (TNBC) subtype. These results suggest that methylation and expression status of PON3 in BC and BC subtypes (TNBC, luminal and HER2) may indicate a poor prognosis. The PON3 gene could be a negative prognostic marker in BC. However, the results should be supported by prospective studies.

Integrated Transcriptome Analysis of Radioresistant Cells Revealed Genes and Pathways Predictive Of Tumor Response to Radiotherapy and Chemotherapy in Breast Cancer

AbstractBreast cancer cells exposed to radiotherapy frequently develop radiation resistance through molecular and phenotypic changes. While there is evidences of pathways controlling radioresistance, the evolution of diverse cell phenotypes and transcriptional changes as mediators of radioresistance in breast cancer are restricted. Moreover, the effectiveness of the chemotherapy on radioresistant cells remains uncertain. In this work, an isogenic model of radioresistant breast cancer cells (RR cells) is used to study this phenotype. RR cells show high survival rates after radiation, moreover, RR cells of the triple negative breast cancer (TNBC) subtype show a significantly advanced invasiveness phenotype. Notably, RR cells are significantly sensitive to chemotherapy by inhibit cell survival and promote apoptosis. Transcriptomics and gene co‐expression network analysis identify differentially expressed genes (DEGs) and hub genes related to survival and apoptosis pathways in the RR cells of luminal subtype, while in TNBC subtype, cell migration, cell differentiation, and immune pathways are enriched. Hub genes predict the failure of radiotherapy in breast cancer patients, but they are also related to pathological complete response after chemotherapy. Transcriptome changes during acquired radioresistance uncover genes and pathways associated to radio and chemotherapy response. These results demonstrate that radioresistant pathways may converge to develop collateral chemo‐sensitivity.

Differential genomic profiling of the progesterone receptor (PR) negative and estrogen receptor (ER) low metastatic breast cancer subtypes through circulating tumor DNA: A TNBC-like disease subtype?

1039 Background: ER positive/HER2 negative metastatic breast cancer (MBC) is typically regarded as a single entity, although significant clinical differences are often observed, especially in the ER low subgroup (ER&lt;10% regardless of PR status). This study aims to investigate genomic differences among HER2 negative subtypes (i.e., ERlow, PRpos [ER &gt;10%/PRpositve], PRneg [ER &gt;10%/PRnegative], triple negative [TNBC]) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS). Methods: The study analyzed a retrospective cohort of 879 patients (pts) with HER2 negative MBC with ctDNA testing using the Guardant360 NGS panel within a large multicenter academic consortium. ER, PR and HER2 status was defined based on the most updated biopsy. Associations across single nucleotide and copy number variations (SNVs and CNVs) and ER/PR status were tested by multinomial logistic regression in terms of Relative Risk Ratio (RRR), adjusting for significant clinical characteristics (i.e., lines of treatment, metastatic sites, histology). Oncogenic pathway was defined based on reference (1). Prognosis was analyzed through Cox regression for overall survival (OS) defined from time of ctDNA collection. Results: Among the 879 analyzed pts, PRpos was the most prevalent subtype (N:420, 48%), followed by PRneg (N:216, 24%), TNBC (N:209, 24%), and ERlow (N:34, 4%). Compared to PRpos, lobular MBC was less common in TNBC (RRR: 0.33, P=0.001), while lung involvement was more frequent in TNBC (RRR: 1.53, P=0.019), and liver involvement in PRneg (RRR: 1.44, P=0.035). In contrast, bone metastases were less common in TNBC and ERlow (RRR: 0.26, P&lt;0.001 and RRR: 0.45, P=0.03, respectively). After multivariable analysis, ERlow and TNBC were associated with a higher incidence of TP53 SNVs (RRR: 5.42, P&lt;0.001 and RRR: 3.08, P=0.007, respectively) and a lower incidence of ESR1 SNVs (RRR: 0.23, P=0.025 and not detected, respectively). TNBC exhibited significantly fewer PIK3CA SNVs (RRR: 0.41, P=0.005) and CCND1 CNVs (RRR: 0.20, P=0.009), along with a higher frequency of CCNE1 CNVs (RRR: 6.47, P&lt;0.001). PRneg had a lower incidence of ESR1 SNVs (RRR: 0.52, P=0.013) and more frequent FGFR1 CNVs (RRR: 2.61, P=0.004). Similar results were observed across oncogenic pathways. Compared to PRpos, OS analysis indicated poorer outcomes across all subgroups (PRneg HR: 1.29 P = 0.035, TNBC HR: 1.96 P&lt;0.001, ERlow HR: 2.32 P&lt;0.001). Conclusions: This study highlights several clinical and molecular similarities between TNBC and ERlow MBC, while suggesting specific endocrine resistance mechanisms in PRneg that are less reliant on ESR1 and more exposed to alternative mechanisms such as FGFR1 amplification. These findings underscore the importance of more precise disease subtyping to inform future drug development trials. 1. Sanchez-Vega F et al, Cell. 2018.

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p &amp;lt; 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p &amp;lt; 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR &amp;lt; 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p &amp;lt; 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p &amp;lt; 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p &amp;lt; 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-09.

Abstract 211: Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer

Abstract Metastatic Breast Cancer is accountable for over half a million deaths worldwide each year, the majority of which are from the triple negative breast cancer (TNBC) subtype, known to be highly aggressive, metastatic and resistant to targeted therapy. Metastasis is driven by the ability of cancer cells to undergo epithelial to mesenchymal transition (EMT), a cell state transition involving a gain of stemness, cell motility and chemoresistance. EMT has been well characterized using a range of techniques based upon quantitative proteomics and transcription profiling, yet targeting EMT to prevent tumor metastasis and chemoresistance is an obstacle that remains to be fully overcome. Using a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS), we have measured changes in protein stability upon induction of EMT and have uncovered key enzymes in the citric acid (TCA) cycle that are stabilized during EMT of human mammary epithelial cells. Such enzymes are responsible for the conversion of TCA cycle intermediates required for a recently discovered post-translational modification. In TNBC cells, we find that loss of these enzymes causes not only global changes to crucial post-translational modification of mitochondrial proteins, but very specific alterations in several cytoskeletal proteins, leading to altered EMT status as well as reduced tumor growth and initiation both in vitro and in vivo. This demonstrates the importance of TCA cycle-controlled post-translational modification of cytoskeletal proteins in tumorigenesis and establishes basis for further investigation into their role in EMT and metastasis of breast cancer. Through pharmacological inhibition and immunofluorescence imaging, we discovered regulators of post-translational modifications that may be viable drug targets to overcome chemoresistance and relapse in TNBC patients with limited and inefficacious options. Citation Format: Jessica A. Lidster, Ser Yue Loo, Zhengwei Wu, Lingyun Dai, Seow Qi Ng, Nayana Prabhu, Dennis Kappei, Wai Leong Tam. Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer [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 211.

TNF-α induced NF-κB mediated LYRM7 expression modulates the tumor growth and metastatic ability in breast cancer

Tumor microenvironment (TME) of solid tumors including breast cancer is complex and contains a distinct cytokine pattern including TNF-α, which determines the progression and metastasis of breast tumors. The metastatic potential of triple negative breast cancer subtypes is high as compared to other subtypes of breast cancer. NF-κB is key transcription factor regulating inflammation and mitochondrial bioenergetics including oxidative phosphorylation (OXPHOS) genes which determine its oxidative capacity and generating reducing equivalents for synthesis of key metabolites for proliferating breast cancer cells. The differential metabolic adaptation and OXPHOS function of breast cancer subtypes in inflammatory conditions and its contribution to metastasis is not well understood. Here we demonstrated that different subunits of NF-κB are differentially expressed in subtypes of breast cancer patients. RELA, one of the major subunits in regulation of the NF-κB pathway is positively correlated with high level of TNF-α in breast cancer patients. TNF-α induced NF-κB regulates the expression of LYRM7, an assembly factor for mitochondrial complex III. Downregulation of LYRM7 in MDA-MB-231 cells decreases mitochondrial super complex assembly and enhances ROS levels, which increases the invasion and migration potential of these cells. Further, in vivo studies using Infliximab, a monoclonal antibody against TNF-α showed decreased expression of LYRM7 in tumor tissue. Large scale breast cancer databases and human patient samples revealed that LYRM7 levels decreased in triple negative breast cancer patients compared to other subtypes and is determinant of survival outcome in patients. Our results indicate that TNF-α induced NF-κB is a critical regulator of LYRM7, a major factor for modulating mitochondrial functions under inflammatory conditions, which determines growth and survival of breast cancer cells.

Abstract B086: Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models

Abstract Background: Triple negative breast cancer subtype is associated with poor prognosis and is mostly treated with chemotherapy, including the DNA crosslinking agent Carboplatin. Carboplatin treatment leads to DNA damage and a cell cycle inhibition allowing DNA damage repair. However, many patients are resistant or develop resistance to Carboplatin. The identification of novel treatments against chemoresistant TNBC is a major unmet clinical need. Method: TNBC patient-derived cell lines (PDCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors were used for shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. Validation of this target was performed genetically by shRNA silencing and pharmacologically with commercially available target inhibitors both in vitro and in vivo, using Alamar Blue viability assays and calculating synergy using the Chou Talalay method. DNA damage was assessed using yH2AX expression, and apoptosis using c-PARP and Annexin V, by confocal microscopy, flow cytometry and/or western blotting. Results: We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened and validated it with an ATR shRNA. We tested the combination of the ATR inhibitor (ATRi), Elimusertib, with Carboplatin and found the combination to be synergistic (Average CI=0.30). DNA damage was markedly increased with combination treatment and a pan-nuclear yH2AX staining together with increased pHH3 expression were observed, suggestive of mitotic catastrophe. Cells treated with Carboplatin accumulated in S phase and a shift toward G2/M when Elimusertib was added. Apoptosis was significantly increased with combination treatment. PKMYT1 and WEE1 are two kinases regulating cell cycle entry by inhibiting CDK1, which controls entry into mitosis. Carboplatin treatment resulted in an increase in PKMYT1 expression. PKMYT1 inhibition with RP6306 re-sensitized the cells to Carboplatin more than WEE1 inhibition with Adavosertib, and this synergy was further increased with low doses of Elimusertib. Gene silencing of WEE1 and PKMYT1 both re-sensitized the cells to Carboplatin, but only WEE1 silencing alone significantly prevent proliferation, suggesting the requirement of PKMYT1 activity for resistance but not for cell proliferation without Carboplatin. In vivo validation using the PDXs from which the cell lines have been derived, demonstrated a significant delayed in tumor growth and prolonged survival when combining Carboplatin with ATRi compared to Carboplatin alone in two models. Conclusion: ATR inhibition resensitizes drug resistant TNBC cells to Carboplatin both in vitro and in vivo. The combination of Carboplatin with ATRi led to DNA damage and mitotic catastrophe. PKMYT1 plays a role in Carboplatin-resistant TNBC, collaborating with ATR in managing the cell cycle response to DNA damage, and PKMYT1 inhibition also re-sensitizes cells to Carboplatin, an effect increased with the triple combination of adding ATR inhibition. Citation Format: Juliet Guay, Catherine Chabot, Cédric Darini, Marguerite Buchanan, Adriana Aguilar-Mahecha, Tim Kong, Connie Yang, Mark Basik, Sidong Huang. Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B086.

Does triple negative breast cancer subtype carry poorer prognosis as compared to non-triple negative breast cancer lesion - A study from a tertiary hospital

Background: The breast cancer (BC), in spite of tremendous advances has continue to remain an enigma. The major change in understanding of BC has taken place after understanding of molecular biology. Aims and Objectives: The objectives are as follows:(1) To categorize the incidence of triple negative BC (TNBC) in hospital based data. (2) To study the prognostic outcomes of TNBC. Materials and Methods: The study was an observational prospective for the period of 18th months, from February 2014 to August 2015, where total 146 cases were selected. The data regarding age, sex, tumor size, stage, grade, nodal status, Nottingham prognostic index (NPI), morphology was noted. All patients underwent immunohistochemistry (IHC) study, maintaining higher quality assurance in the same IHC laboratory. Results: Our study revealed that out of 146 patients, 71 patients are TNBC. The mean age of presentation was 54.06 years in TNBC group. About 69.0% patients presented with Grade III tumor and 74.6% patient presented with NPI ≥5.4 among TNBC. However, 56% patients presented with NPI ≥5.4 among NON TNBC. Most of the morphological variant was IDC (95.8%), 85.9% patients presented with only lymph nodes metastasis among TNBC. Conclusion: The study is tremendously significant from Indian perspective. It proofs that TNBC had the point of diagnosis, seems to be more aggressive. It suggests that Indian BC is likely to be younger at presentation as compared to its Western counterpart.

This house believes that: MARI/TAD is better than sentinel node biopsy after PST for cN+ patients.

The increasing use and effectiveness of primary systemic treatment (PST) enables tailored locoregional treatment. About one third of clinically node positive (cN+) breast cancer patients achieve pathologic complete response (pCR) of the axilla, with higher rates observed in Human Epidermal growth factor Receptor (HER)2-positive or triple negative (TN) breast cancer subtypes. Tailoring axillary treatment for patients with axillary pCR is necessary, as they are unlikely to benefit from axillary lymph node dissection (ALND), but may suffer complications and long-term morbidity such as lymphedema and impaired shoulder motion. By combining pre-PST and post-PST axillary staging techniques, ALND can be omitted in most cN+patients with pCR. Different post-PST staging techniques (MARI/TAD/SN) show low or ultra-low false negative rates for detection of residual disease. More importantly, trials using the MARI (Marking Axillary lymph nodes with Radioactive Iodine seeds) procedure or sentinel lymph node biopsy (SLNB) as axillary staging technique post-PST have already shown the safety of tailoring axillary treatment in patients with an excellent response. Tailored axillary treatment using the MARI procedure in stage I-III breast cancer resulted in 80% reduction of ALND and excellent five-year axillary recurrence free interval (aRFI) of 97%. Similar oncologic outcomes were seen for post-SLNB in stage I-II patients. The MARI technique requires only one invasive procedure pre-NST and a median of one node is removed post-PST, whereas for the SLNB and TAD techniques two to four nodes are removed. A disadvantage of the MARI technique is its use of radioactive iodine, which is subject to extensive regulations.

Phenotypic heterogeneity drives differential disease outcome in a mouse model of triple negative breast cancer.

The triple negative breast cancer (TNBC) subtype is one of the most aggressive forms of breast cancer that has poor clinical outcome and is an unmet clinical challenge. Accumulating evidence suggests that intratumoral heterogeneity or the presence of phenotypically distinct cell populations within a tumor play a crucial role in chemoresistance, tumor progression and metastasis. An increased understanding of the molecular regulators of intratumoral heterogeneity is crucial to the development of effective therapeutic strategies in TNBC. To this end, we used an unbiased approach to identify a molecular mediator of intratumoral heterogeneity in breast cancer by isolating two tumor cell populations (T1 and T2) from the 4T1 TNBC model. Phenotypic characterization revealed that the cells are different in terms of their morphology, proliferation and self-renewal ability in vitro as well as primary tumor formation and metastatic potential in vivo. Bioinformatic analysis followed by Kaplan Meier survival analysis in TNBC patients identified Metastasis associated colon cancer 1 (Macc1) as one of the top candidate genes mediating the aggressive phenotype in the T1 tumor cells. The role of Macc1 in regulating the proliferative phenotype was validated and taken forward in a therapeutic context with Lovastatin, a small molecule transcriptional inhibitor of Macc1 to target the T1 cell population. This study increases our understanding of the molecular underpinnings of intratumoral heterogeneity in breast cancer that is critical to improve the treatment of women currently living with the highly aggressive TNBC subtype.

Expression of Programmed Death Ligand 1 (PD-L1) in Breast Cancer patients in India and its Correlation with Prognostic Parameters

Background: The PD-1/PD-L1 pathway represents an adaptive immune resistance mechanism that is exerted by tumor cells. The study was conducted to evaluate the expression of PD-L1 in breast carcinoma in India and to find out its correlation with prognostic parameters. Despite numerous studies, there is a lack of literature for such studies in Indian patients. Moreover, the results obtained from these studies have not been uniform. Methods: A hospital-based cross-sectional study was conducted on 150 cases of breast carcinoma. The invasive cancer specimens were assessed for routine microscopy and classified into various histopathological subtypes. Bloom Richardson grading was done. Immunohistochemistry for surrogate molecular classification as well as PD-L1 was performed. PD-L1 expression was then compared with several prognostic parameters such as tumor subtype, tumor grade, surrogate molecular classification and pTNM stage. Data analysis was done using Statistical Package for Social Sciences (SPSS) version 21.0. A P value of &lt;0.05 was considered statistically significant. Results: PD-L1 was positive in 14.67% of patients with score 1 in 6% and score 2 in 8.67% of patients. The PD-L1 expression showed a positive correlation with the tumor of higher grades (grade3). It was significantly higher among IBC with medullary features as compared to IBC-NST, IBC with papillary features and the Lobular type. PD-L1 showed a significant association with surrogate molecular classification as well. Its expression was found to be the highest in Triple negative breast cancer subtype as compared to tumors showing ER/PR positivity (p&lt;0.05). However, there was no significant association between PD-L1 and TNM staging. Conclusion: This study revealed a significant association between PD-L1 several prognostic factors such as higher tumor grade (grade3), Triple Negative breast cancer, and IBC with the medullary pattern subtype. The association between PD-L1 and such prognostic parameters signifies its role in tumor mechanism and makes it a potential target for immunotherapy especially in Triple Negative breast cancer cases which lack specific targeted therapies. Moreover, paucity of literature on PD-L1 in breast cancer patients in India and the data showing conflicting results make this study valuable. Our study shall be helpful in further adding to the knowledge of PD-L1 expression in breast cancer especially in Indian women.

Abstract 1238: Loss of HSPD1 induces cellular plasticity in triple-negative breast cancer cells

Abstract Abstract Background Unlike Breast cancer (BRCA) luminal subtypes, Triple negative breast cancer (TNBC) subtype lacks expression of estrogen receptor-α (ERα/ESR1), progesterone receptor (PGR) and/or epidermal growth factor (EGF) family receptor HER2/ERBB2 where patients do not benefit from endocrine and HER2 based therapies in addition to its poor differentiation that results in greater aggressiveness and poorer prognosis. The 60 KD Heat shock protein (HSP60) is a Mitochondrial protein that plays a role in stabilizing and refolding proteins and is regulated by HSPD1 gene. In normal cells, HSPD1 gene expression is controlled, however in TNBC, HSPD1 overexpression is common and is associated with poor cancer-specific survival. This study aims to understand downstream signaling pathways dependent on the function of HSPD1 in TNBC. Method To understand downstream signaling pathways dependent on the function of HSPD1, we analyzed the transcriptomic profiles of metastatic MDA-MB-231-WT and HSPD1-KD cells. Results Bioinformatics data analysis shows that HSPD1 is overexpressed in TNBC, particularly in basal subtype. High expression of HSPD1 related to low survival in breast cancer patients. HSPD1 KD in MDA-MB-231 cells identified several differentially regulated genes (DEG) as compared to WT MDA-MB-231 cells by RNA-seq analysis. Pathway analysis of DEG showed downregulation of mitochondrial respiration, thermogenesis, citric acid cycle, PD-1 signaling while upregulation of estrogen dependent gene expression, estrogen mediated signaling, nuclear receptor transcription pathway. These suggests that HSP60 overexpression in TNBC promotes basal type phenotype. Downregulation of HSP60 expression upregulated hormone receptors, particularly estrogen receptor (ESR1) and androgen receptor (AR) expression, thus inducing basal to luminal switch in MDA-MB-231 cells. These findings suggest that loss of HSPD1 significantly Induces plasticity in TNBC. Conclusion Our results suggest that loss of HSPDI in the context TNBC cells may promotes oncogenic transformation to an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the upregulation of Estrogen-receptors signaling pathways. Ongoing analyses will be performed to identify the possible links between loss of HSPD1 and key transcription factors that define mammary epithelial cell differentiation. Citation Format: Muhammad G. Khodary, Alehegne W. Yirsaw, Temesgen Samuel, Clayton Yates, Deepa Bedi. Loss of HSPD1 induces cellular plasticity in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1238.

Abstract 5492: Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor

Abstract Introduction: Triple negative breast cancer (TNBC) subtype of breast cancer, which lacks molecular markers such as HER2 and estrogen/progesterone receptors, has limited treatment options. Exosomes are nano-sized (30-150 nm) spherical vesicles, derived from the endosomal system for intercellular communications, can be engineered to express targeting peptides to target specifically CD206 positive M2 macrophage. Investigators have used either synthetic nanoparticles or fusion protein to deliver Fc-IgG2b to initiate Antibody dependent cellular cytotoxicity (ADCC) but reports are showing a lack of ADCC following tagging with gold nanoparticles. To overcome this, in our lab, we developed engineered exosomes in non-tumorous cells, HEK293, to carry therapeutic Fc-mIgG2b to enhance ADCC and to carry peptide to deplete CD206+ M2 macrophages. We investigated the effectiveness of engineered therapeutic exosomes to target and deplete immunosuppressive CD206+ M2 macrophages in TNBC in combination with anti PD-1. Methods: In this study, we developed syngeneic metastatic TBNC model (luciferase + 4T1 cells in Balb/c) by implantation of 50K 4T1-luci cells in fat pad. On day 1 of tumor implantation, animals were treated with vehicle, engineered exosomes, anti-PD1 plus engineered exosomes, 2 dose/week for 3 weeks. They were euthanized 1 day after the last dose of treatment. Single-cell suspension was made from the primary TME, lung, and spleen. Using flow cytometry surfaces markers were investigated to determine different T-cell and myeloid cell populations. Results: We observed very surprising results following three weeks of therapies. Although the number of cytotoxic T-cells was significantly higher in the primary and metastatic TME in exosome-treated animals, there was also an increased number of proliferative T reg cells. The number of CD8+ cells was decreased following concurrent therapy with an anti-PD1 antibody. Most interestingly, lung tissue macrophages (CD11c) showed significantly increased PD-L1 expression following exosomes and anti PD1 with engineered exosomes therapies. Conclusion: This study demonstrates that treatment with engineered therapeutic exosome targeting M2 macrophages with anti PD-1 could be utilized effectively. This study will also make a greater impact in exosome engineering based targeted therapy and will open new arena to target and deplete pathogenic cell in different tumors using engineered exosome. Citation Format: Mahrima Parvin, Mohammad H. Rashid, Ahmet Alptekin, Ali S. Arbab. Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5492.

Abstract 4522: Pathologic complete response rate across triple negative breast cancer subtypes in the IMMUcan study

Abstract Background: IMMUcan (SPECTA NCT02834884) is a European public-private effort to generate molecular and cellular profiling data of the human tumor microenvironment (TME) from up to 3,000 patients, to better understand how the immune system and tumors interact. Triple-negative breast cancer (TNBC) is a heterogenous disease with distinct molecular subtypes characterized by different gene expression, genomic profiles, and clinical outcomes. Here, we explored the association between molecular subtypes and response to neoadjuvant chemotherapy as well as levels of immune cell infiltration in the prospective IMMUcan TNBC cohort. Methods: At the cut-off date on June 29th, 2022, we identified a first cohort of 132 patients to perform preliminary analyses, of which 109 had pre-treatment RNAseq data available. Among those, pathological Complete Response (pCR) information was available for a total of 86 patients. Preliminary data included the density of CD3+, CD20+ and CD8+ immune cells from the stroma and tumor compartments from multiplex-immunofluorescence (mIF) for 69 patients. TNBC subtypes were computed as described by Bareche et al. Association of continuous variables with pCR was evaluated with logistic regression (univariate analysis). Results: The pCR rates differed across TNBC subtypes (Fisher’s test, P = 0.02). In detail, pCR was achieved in 88% (8/9) of basal like (BL), 63% (17/27) of immunomodulatory (IM) and 45% (16/35) of mesenchymal (M) tumors, while lower pCR rates were observed for the mesenchymal stem-like (MSL) and luminal androgen receptor (LAR) subtypes with 37% (3/8) and 14% (1/7), respectively, suggesting differences in the sensitivity to neoadjuvant chemotherapy in line with previous reports. When considered as continuous signature scores, pCR was positively associated to BL levels (OR = 2.7, 95% CI, 1.5-4.8; False Discovery Rate [FDR] = 0.0031), and negatively associated to MSL and LAR signature levels (OR = 0.53, 95% CI, 0.32-0.87; FDR = 0.019; OR = 0.30, 95% CI, 0.12-0.74; FDR= 0.019). In addition, CD3+ cell densities in the tumor and stroma were significantly higher in IM tumors (P = 0.002 and P &amp;lt; 0.001), while lower levels were found in M tumors (P = 0.005, P = 0.002). Similarly, stromal CD20+ levels were higher in IM tumors (P = 0.008) and lower in M tumors (P = 0.006), suggesting a more immunosuppressive TME in the M subtype. CD8+ cells levels were not significantly different between subtypes. Conclusions: These preliminary results show relevant differences in the pCR rates among TNBC subtypes, to be confirmed on a larger series of patients and further suggesting a substantial TNBC heterogeneity regarding treatment response. Of note, preliminary mIF results suggest a differential role of the immune response across molecular subtypes. Additional analyses integrating mIF and genomic data are currently ongoing. Funding: IMI2 JU grant agreement 821558, supported by EU’s Horizon 2020 and EFPIA. Citation Format: Andrea Joaquin Garcia, Mattia Rediti, Abdelkader Benyagoub, Stéphanie Tissot, Sylvie Rusakiewicz, Robin Liechti, Flavia Marzetta, Nicolas Penel, Julio Oliveira, Jean-Charles Goeminne, Pierre Fournel, Andreia Capela, Xiaoxiao Wang, Delphine Vincent, Françoise Rothe, Marie Morfouace, Henoch Hong, Donald Jackson, Christos Sotiriou, Laurence Buisseret. Pathologic complete response rate across triple negative breast cancer subtypes in the IMMUcan study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4522.

Hyperinsulinemic and Pro-Inflammatory Dietary Patterns and Metabolomic Profiles Are Associated with Increased Risk of Total and Site-Specific Cancers among Postmenopausal Women.

We evaluated associations of the Empirical Dietary Index for Hyperinsulinemia (EDIH), Empirical Dietary Inflammatory Pattern (EDIP) and Healthy Eating Index (HEI2015) and their metabolomics profiles with the risk of total and site-specific cancers. We used baseline food frequency questionnaires to calculate dietary scores among 112,468 postmenopausal women in the Women's Health Initiative. We used multivariable-adjusted Cox regression to estimate hazard ratios (HR) and 95% confidence intervals for cancer risk estimation. Metabolomic profile scores were derived using elastic-net regression with leave-one-out cross validation. In over 17.8 years, 18,768 incident invasive cancers were adjudicated. Higher EDIH and EDIP scores were associated with greater total cancer risk, and higher HEI-2015 with lower risk: HRQ5vsQ1(95% CI): EDIH, 1.10 (1.04-1.15); EDIP, 1.08 (1.02-1.15); HEI-2015, 0.93 (0.89-0.98). The multivariable-adjusted incidence rate difference(Q5vsQ1) for total cancer was: +52 (EDIH), +41 (EDIP) and -49 (HEI-2015) per 100,000 person years. All three indices were associated with colorectal cancer, and EDIH and EDIP with endometrial and breast cancer risk. EDIH was further associated with luminal-B, ER-negative and triple negative breast cancer subtypes. Dietary patterns contributing to hyperinsulinemia and inflammation were associated with greater cancer risk, and higher overall dietary quality, with lower risk. The findings warrant the testing of these dietary patterns in clinical trials for cancer prevention among postmenopausal women.