Triple-negative Breast Cancer Recurrence Research Articles

A novel humanized Frizzled-7-targeting antibody enhances antitumor effects of Bevacizumab against triple-negative breast cancer via blocking Wnt/\u03b2-catenin signaling pathway

BackgroundAnti-angiogenic therapy has been widely applied to the clinical treatment of malignant tumors. However, the efficacy of such treatments has been called into question, especially in triple-negative breast cancer (TNBC). Bevacizumab, the first anti-angiogenic agent approved by FDA, actually increases invasive and metastatic properties of TNBC cells, resulting from the activation of Wnt/β-catenin signaling in response to hypoxia. As a critical receptor of Wnt/β-catenin signaling, Frizzled-7 (Fzd7) is aberrantly expressed in TNBC, indicating Fzd7 a potential target for developing drugs to be combined with anti-angiogenic agents.MethodsHybridoma technique and antibody humanization technique were utilized to generate a Fzd7-targeting antibody (SHH002-hu1). Biolayer interferometry (BLI) assay and near infrared (NIR) imaging were conducted to detect the affinity and targeting ability of SHH002-hu1. Next, whether SHH002-hu1 could suppress the invasion and migration of TNBC cells induced by Bevacizumab were validated, and the underlying molecular mechanisms were elucidated by luciferase reporter and western blot assays. The nude-mice transplanted TNBC models were established to assess the anti-TNBC activities of SHH002-hu1 when combined with Bevacizumab. Then, the effects on putative TNBC stem-like cells and Wnt/β-catenin signaling were evaluated by immunofluorescence (IF). Further, the tumor-initiating and self-renew capacity of TNBC cells were studied by secondary nude mouse xenograft model and sphere formation assay. In addition, the effects of SHH002-hu1 on the adaptation of TNBC cells to hypoxia were evaluated by the detection of vasculogenic mimicry (VM) and hypoxia-inducible factor-1α (HIF-1α) transcriptional activity.ResultsThe novel humanized antibody targeting Fzd7 (SHH002-hu1) exhibited extremely high affinity with Fzd7, and specifically targeted to Fzd7+ cells and tumor tissues. SHH002-hu1 repressed invasion, migration and epithelial-mesenchymal cell transformation (EMT) of TNBC cells induced by Bevacizumab through abating Wnt/β-catenin signaling. SHH002-hu1 significantly enhanced the capacity of Bevacizumab to inhibit the growth of TNBC via reducing the subpopulation of putative TNBC stem-like cells, further attenuating Bevacizumab-enhanced tumor-initiating and self-renew capacity of TNBC cells. Moreover, SHH002-hu1 effectively restrained the adaptation of TNBC cells to hypoxia via disrupting Wnt/β-catenin signaling.ConclusionSHH002-hu1 significantly enhances the anti-TNBC capacity of Bevacizumab, and shows the potential of preventing TNBC recurrence, suggesting SHH002-hu1 a good candidate for the synergistic therapy together with Bevacizumab.

Quiescent Cancer Cells Resist T Cell Attack by Forming an Immunosuppressive Niche

Immunotherapy is a promising treatment for Triple-Negative Breast Cancer (TNBC), but patients recur, arising the need to understand mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that survive T-cell attack are in a quiescent state. Quiescent Cancer Cells (QCCs) are found clustering together forming regions with reduced immune infiltration. QCCs display superior tumorigenic capacity and higher expression of stemness genes than their proliferative counterparts. We adapted single-cell-RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside QCC niches. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T-cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their specific intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T-cell function. Eliminating QCCs holds the promise to counteract resistance to immunotherapy and prevent disease recurrence in TNBC.

Regulation of cancer stem cells in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC) is the most lethal subtype of breast cancer. Despite the successes of emerging targeted therapies, relapse, recurrence, and therapy failure rates in TNBC significantly outpace other subtypes of breast cancer. Mounting evidence suggests accumulation of therapy resistant Cancer Stem Cell (CSC) populations within TNBCs contributes to poor clinical outcomes. These CSCs are enriched in TNBC compared to non-TNBC breast cancers. The mechanisms underlying CSC accumulation have been well-characterized and discussed in other reviews. In this review, we focus on TNBC-specific mechanisms that allow the expansion and activity of self-renewing CSCs. We highlight cellular signaling pathways and transcription factors, specifically enriched in TNBC over non-TNBC breast cancer, contributing to stemness. We also analyze publicly available single-cell RNA-seq data from basal breast cancer tumors to highlight the potential of emerging bioinformatic approaches in identifying novel drivers of stemness in TNBC and other cancers.

FZD5 contributes to TNBC proliferation, DNA damage repair and stemness

Chemotherapy currently remains the standard treatment for triple-negative breast cancer (TNBC). However, TNBC frequently develop chemoresistance, which is responsible for cancer recurrence and distal metastasis. Both DNA damage repair and stemness are related to chemoresistance. FZD5, a member in Frizzled family, was identified to be preferentially expressed in TNBC, and associated with unfavorable prognosis. Loss and gain of function studies revealed that FZD5 contributed to TNBC cell G1/S transition, DNA replication, DNA damage repair, survival, and stemness. Mechanistically, transcription factor FOXM1, which promoted BRCA1 and BIRC5 transcription, acted as a downstream effecter of FZD5 signaling. FOXM1 overexpression in FZD5-deficient/low TNBC cells induced FZD5-associated phenotype. Finally, Wnt7B, a specific ligand for FZD5, was shown to be involved in cell proliferation, DNA damage repair, and stemness. Taken together, FZD5 is a novel target for the development of therapeutic strategies to overcome chemoresistance and prevent recurrence in TNBC.

A “dual-guide” bioinspired drug delivery strategy of a macrophage-based carrier against postoperative triple-negative breast cancer recurrence

Recurrence after tumor resection is mainly caused by post-operative inflammation and residual cancer cells, which is a serious obstacle to breast cancer treatment. Traditional nanoparticles rely primarily on the enhanced permeability and retention (EPR) effect in well-vascularized tumors. In this study, a macrophage-based carrier is designed to enhance the efficiency of targeting to recurrent tumors through a “dual-guide” strategy. After tumor resection, a burst of inflammatory factors occurs in the resection wound, which can recruit monocytes/macrophages rapidly. Combined with the tropism of monocyte chemoattractant protein, a large number of macrophage-mediated carriers will be recruited to surgical recurrence sites. Octaarginine (RRRRRRRR, R8)-modified liposomes in macrophages contain two agents with different pharmacological mechanisms, paclitaxel (PTX) and resveratrol (Res), which have enhanced therapeutic effects. In vitro study demonstrated that macrophage-mediated carriers approach 4 T1 cells through an inflammatory gradient and reach recurrence tumors through a “dual-guide” strategy. Then, membrane fusion and inflammation-triggered release deliver the drug into the recurrent tumor cells. In vivo experiments show that macrophage-based carriers exhibit effective tumor-targeting ability, especially in post-operation situations. More importantly, macrophage-mediated liposomes encapsulated with PTX and Res inhibit tumor recurrence in both ectopic and orthotopic 4 T1 post-operative recurrence models.

An Intelligent Biomimetic Nanoplatform for Holistic Treatment of Metastatic Triple-Negative Breast Cancer via Photothermal Ablation and Immune Remodeling.

Metastasis is one of the main causes of failure in the treatment of triple-negative breast cancer (TNBC). Immunotherapy brings hope and opportunity to solve this challenge, while its clinical applications are greatly inhibited by the tumor immunosuppressive environment. Here, an intelligent biomimetic nanoplatform was designed based on dendritic large-pore mesoporous silica nanoparticles (DLMSNs) for suppressing metastatic TNBC by combining photothermal ablation and immune remodeling. Taking advantage of the ordered large-pore structure and easily chemically modified property of DLMSNs, the copper sulfide (CuS) nanoparticles with high photothermal conversion efficiency were in situ deposited inside the large pores of DLMSNs, and the immune adjuvant resiquimod (R848) was loaded controllably. A homogenous cancer cell membrane was coated on the surfaces of these DLMSNs, followed by conjugation with the anti-PD-1 peptide AUNP-12 through a polyethylene glycol linker with an acid-labile benzoic-imine bond. The thus-obtained AM@DLMSN@CuS/R848 was applied to holistically treat metastatic TNBC in vitro and in vivo. The data showed that AM@DLMSN@CuS/R848 had a high TNBC-targeting ability and induced efficient photothermal ablation on primary TNBC tumors under 980 nm laser irradiation. Tumor antigens thus generated and increasingly released R848 by response to the photothermal effect, combined with AUNP-12 detached from AM@DLMSN@CuS/R848 in the weakly acidic tumor microenvironment, synergistically exerted tumor vaccination, and T lymphocyte activation functions on immune remodeling to prevent TNBC recurrence and metastasis. Taken together, this study provides an intelligent biomimetic nanoplatform to enhance therapeutic outcomes in metastatic TNBC.

Cytoplasmic CCR7 (CCR7c) immunoexpression is associated with local tumor recurrence in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of cancer, which tests negative for estrogen receptors, progesterone receptors, and lacks overexpression of the human epidermal growth factor 2 (C-erbB2, HER2/neu) gene. The expression of chemokines and their receptors, including CCR7, has been described in several types of cancer, contributing to tumor progression. This study investigated the association between the membrane and cytoplasmic CCR7 expression and the prognosis of TNBC. Surgical paraffin histopathology blocks and clinico-pathological data were assessed from 133 patients. Samples were analyzed by immunohistochemistry and immunofluorescence using the Tissue Microarray technique for scoring the intensity of CCR7 expression. TNBC patients in which the CCR7 labeling was predominantly in the cytoplasm of tumor cells presented increased local tumor recurrence (P = 0.033). Conversely, there was no statistical difference in five-year overall survival between the patients with low (77%) versus high (80%) cytoplasmic CCR7 expression (P = 0.7104). Additionally, the risk of death between these groups was 1.19 (95% CI = 0.48-2.91). The cytoplasmic CCR7 expression associates with an increased incidence of tumor relapse in TNBC, not affecting patients survival. Consequently, the cell compartment in which the CCR7 localizes could serve as a prognostic marker in this cancer subtype.

DR-5 and DLL-4 mAb Functionalized SLNs of Gamma-Secretase Inhibitors- An Approach for TNBC Treatment.

Triple-negative breast cancer (TNBC) is the most aggressive and heterogeneous cancer subtypes. High rates of metastasis, poor prognosis, and drug resistance are the major problems associated with TNBC. The current chemotherapeutics eliminate only the bulk tumor cells (non-BCSCs) and do not affect breast cancer stem cells (BCSCs). The BCSCs which are left behind after chemotherapy is reported to promote recurrence and metastasis of TNBC. Death receptor-5 (DR-5) is exclusively expressed in TNBCs and mediates the extrinsic pathway of apoptosis. DR-5, therefore, can be exploited for targeted drug delivery and to induce apoptosis. Gamma-secretase mediated Notch signaling in BCSCs regulates its proliferation, differentiation, and metastasis. The endogenous ligand, Delta-like ligand 4 (DLL4), is reported to activate this Notch signaling in TNBC. Blocking this signaling pathway using both gamma-secretase inhibitors (GSIs) and DLL4 monoclonal antibody (mAb) may produce synergistic benefits. Further, the GSIs (DAPT, LY-411575, RO4929097, MK0752, etc.) suffer from poor bioavailability and off-target side effects such as diarrhea, suppression of lymphopoiesis, headache, hypertension, fatigue, and ventricular dysfunctions. In this hypothesis, we discuss Solid lipid nanoparticles (SLNs) based drug delivery systems containing GSIs and surface modified with DR-5 and DLL4 monoclonal antibodies (mAb) to effectivity target and treat TNBC. The delivery system is designed to deliver the drug cargo precisely to TNBCs through its DR-5 receptors and hence expected to reduce the off-target side effects of GSIs. Further, DLL4 mAb and GSIs are expected to act synergistically to block the Notch signal mediated BCSCs proliferation, differentiation, and metastasis.

Prognostication of a 13-immune-related-gene signature in patients with early triple-negative breast cancer.

We investigated the expression profiles of immune genes in patients with triple-negative breast cancer (TNBC) to identify the prognostic value of immune genes and their clinical implications. NanoString nCounter Analysis of 770 immune-related genes was used to measure immune gene expression in patients with TNBC who underwent curative surgery followed by adjuvant chemotherapy at Samsung Medical Center between 2000 and 2004. Statistical analyses were conducted to identify the associations between gene expression and distant recurrence-free survival (DRFS). Of 1189 patients who underwent curative BC surgery, 200 TNBC patients were included and stage was the only clinical factor predictive of DRFS. In terms of immune genes, 155 of 770 genes were associated with DRFS (p < 0.01). Further multivariate analysis revealed that 13 genes, CD1B, CD53, CT45A1, GTF3C1, IL11RA, IL1RN, LRRN3, MAPK1, NEFL, PRKCE, PTPRC, SPACA3 and TNFSF11, were associated with patient prognosis (p < 0.05). The prognostic value of stage and expression levels of 13 immune genes was analyzed and the area under the receiver operating characteristic curve (AUC) was 0.923. Based on the AUC, we divided patients into three genetic risk groups and DRFS rate was significantly different according to genetic risk groups, even in the same stage (p < 0.001). In this study, a 13-gene expression profile in combination with stage precisely predicted distant recurrence of early TNBC. Therefore, this 13-immune-gene signature could help predict TNBC prognosis and provide guidance for treatment as well as the opportunity to develop new targets for immunotherapy in TNBC patients.

Triple-negative and non–triple-negative breast cancer in a south Indian cohort.

e13110 Background: The heterogeneity of breast cancer explains in part the differences in the morbidity and mortality of this disease. Triple-negative breast cancer (TNBC) is a specific subset of tumors characterized by the absence of the 3 most commonly targeted biomarkers considered for breast cancer treatment: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). TNBC accounts for 15% to 20% of all breast cancer diagnoses and usually has a more aggressive clinical course, with worse evolution within the first 3 to 5 years after diagnosis; early and higher rates of distant recurrences, typically visceral; and poor survival. Methods: To analyze the clinical, pathological, and sociodemographic aspects between triple-negative breast cancer (TNBC) and non-TNBC in a Kerala cohort and identify potential prognostic factors. This hospital-based retrospective cohort study (From January 2017 till December 2018) included 465 women with breast cancer treated at MVR Cancer Centre &amp; Research Institute in North Kerala. Overall and disease-free survival was compared; prognostic factors were evaluated. Results: Triple-negative breast cancer corresponded to 16.3% of breast cancer diagnosis and was more prevalent among rural women. The patients with TNBC tended to present with stage III cancer, high p53 expression, lymphocytic infiltration, and multifocality and treated with radical surgery and chemotherapy. The 2-year overall and disease-free survival were 52.1% and 43.5% for TNBC and 83.8% and 73.4% for non-TNBC, respectively ( P &lt; .001). The TNBC recurrence was associated with multicentricity, whereas lymph node involvement increased the risk of both recurrence and death. Non-TNBC worse clinical course was associated with rural women, younger age, lymph node involvement, and advanced stage. Conclusions: Triple-negative breast cancer exhibited a more aggressive behavior, earlier and more frequent recurrence, and worse survival compared with non-TNBC. While biological and social variables were associated with poorer prognosis in non-TNBC, only lymph node involvement and multicentricity were correlated with worse clinical outcomes in TNBC.

MEGF11 is related to tumour recurrence in triple negative breast cancer via chemokine upregulation

Our previous study demonstrated that upregulation of multiple epidermal growth factor-like domains 11 (MEGF11) gene expression is involved in the mechanism by which recurrence of Triple Negative Breast Cancer (TNBC) occurs. Our aim was to elucidate the role of MEGF11 expression in TNBC cells, both in vitro and in vivo, and in human tissue. Following MEGF11 gene knockdown (∆MEGF11) or over-expression in MDA-MB-231 and MB-468 cells, cell growth and chemokine gene expression were evaluated. In vivo, tumour growth of implanted human TNBC cells and the number of circulating 4T1 mouse tumour cells were measured. There was a significant decrease in cell growth via inhibition of AKT, NF-kB, CREB and AP-1 activation in ∆MEGF11 MDA-MB-231 and 468 cells. This also resulted, in vivo, in a suppression of tumour growth and a decrease in the number of mouse circulating 4T1 breast cancer cells. Surprisingly, overexpression of MEGF11 upregulated the expression of various chemokines and proinflammatory cytokines via AKT activation, but there was no increase in cell proliferation. MEGF11 was found to cross-talk positively with IL-17A signalling. Patients with tumours that over-expressed MEGF11 had a poorer prognosis. We conclude that MEGF11 plays an important role in tumour survival and that overexpression of MEGF11 induces both a cytokine and a chemokine cascade, which will favour the tumour microenvironment in terms of distant metastasis. MEGF11 might be a potential therapeutic target for preventing TNBC recurrence.

Three-dimensional radiomics of triple-negative breast cancer: Prediction of systemic recurrence

This paper evaluated 3-dimensional radiomics features of breast magnetic resonance imaging (MRI) as prognostic factors for predicting systemic recurrence in triple-negative breast cancer (TNBC) and validated the results with a different MRI scanner. The Rad score was generated from 3-dimensional radiomic features of MRI for 231 TNBCs (training set (GE scanner), n = 182; validation set (Philips scanner), n = 49). The Clinical and Rad models to predict systemic recurrence were built up and the models were externally validated. In the training set, the Rad score was significantly higher in the group with systemic recurrence (median, −8.430) than the group without (median, −9.873, P < 0.001). The C-index of the Rad model to predict systemic recurrence in the training set was 0.97, which was significantly higher than in the Clinical model (0.879; P = 0.009). When the models were externally validated, the C-index of the Rad model was 0.848, lower than the 0.939 of the Clinical model, although the difference was not statistically significant (P = 0.100). The Rad model for predicting systemic recurrence in TNBC showed a significantly higher C-index than the Clinical model. However, external validation with a different MRI scanner did not show the Rad model to be superior over the Clinical model.

Abstract P3-08-45: Pattern and biomarkers of recurrence in 305 triple negative breast cancer patients treated in a French comprehensive cancer center

Abstract Background: Triple negative breast cancer (TNBC) is the most heterogeneous subtype of breast cancer. However, the initial treatment of this disease is still dichotomous: 1st-line surgery (Surg1) followed by adjuvant chemotherapy, or neoadjuvant chemotherapy (NACT) followed by surgery. After radiotherapy, most of the patients (pts) are only observed until recurrence. Our team has pointed earlier (SABCS 2017) that some TNBCs recur very rapidly, in less than 12 months (mo) after NACT. Pts with those tumors have a very poor prognosis (overall survival &amp;lt; 6 mo). On the other side, a recent study has revealed a TNBC subgroup with late recurrences (&amp;gt; 5 yrs post-diagnosis, Rueda et al, Nature 2019). Neither of those groups is currently recognized in clinical practice nor specifically managed. In this study, we analyzed pattern and potential clinico-pathological biomarkers of recurrence in a large TNBC patient cohort treated at a specialized cancer center in France, to get a better insight into the need and possibilities for specific prevention of recurrent TNBC. Pts and methods: The study retrospectively included 305 pts, treated between 2000 and 2015 in Centre Jean Perrin, Clermont-Ferrand (stage I-III: 135 pts by Surg1; 146 pts by NACT; stage IV: 24 pts). The following putative biomarkers were evaluated: breast tumor size, number of involved nodes, tumor histograde, presence of lympho-vascular invasion (LVI), tumor-infiltrating lymphocytes (TILs, according to PMIDs 25214542, 29024776), presence of epithelial-mesenchymal transition (expression of Zeb1 protein by immunohistochemistry) - all at the Surg1 and post-NACT residual tumor (RT), as well as pathological complete response to NACT (pCR). Median follow-up of the pts was 4.8 yrs. The biomarkers were evaluated by univariate and multivariate analysis. Results: Recurrence-free survival (RFS) rate was significantly higher in Surg1 than in NACT cohort (76% vs 62%, resp., p=0.016). Early recurrence rate (RR) (&amp;lt; 12 mo post-Dg) was higher in the latter (5.6%) than in the former (2.4%), whereas the standard and the late RRs were comparable (RR 1-5 yrs post-Dg: 21.4% vs. 32.5%, p=0.05; &amp;gt; 5 yrs: 7.0% vs 6.7%, Surg1 vs NACT, resp.) In Surg1 cohort, the independent recurrence biomarkers were pN (pN0 vs pN+, p=0.003) and LVI+ (LVI+ vs LVI-, p=0.005). In NACT cohort, the only independent recurrence biomarkers were post-NACT RT size (p=0.009) and number of the involved nodes (LN) (p&amp;lt;10-7). When RT size and LN were combined, we obtained 5 post-NACT categories: RT≤1cm/LN=0, n=51; RT&amp;gt;1cm≤2cm/LN=0, n=36; any RT/LN=1, n=18; RT≤2cm/LN≥2, n=11; RT&amp;gt;2cm/LN≥2, n=16, with significantly different 5-year RFS rates (92%, 62%, 54%, 30%, 13%, p&amp;lt;10-7). No predictive biomarker of early or late recurrences was identified, however, we observed a tendency of pts recurring early after NACT to have large RTs (&amp;gt;2cm), with median TILs &amp;lt;5%, in comparison with other non-pCR cases (p=0.10). Interestingly, both the post-NACT early recurring tumors (n=8) and the stage IV at Dg tumors (n=24) had very low TILs (&amp;lt;5%), 8/8 (100%) in the former and 17/20 (85%) in the latter. Details of the TIL analysis will be presented at the meeting. Conclusion: Our results confirm that most TNBC recurrences occur 1-5 yrs post-Dg, however early (&amp;lt; 1yr) and late (&amp;gt; 5yrs) recurrences exist too, in up to 15% pts. The early recurrences are more frequent in the pts treated by NACT, and reflect high baseline metastatic capacity of the tumors. Big post-NACT RT size (&amp;gt; 2 cm), TILs &amp;lt; 5% and ≥ 2 LN+ are worth validation on larger cohorts as biomarkers of TNBC early and standard (1-5 yrs) recurrence. These 3 parameters, easily assessable in routine clinical practice, could help selecting pts with high risk of recurrence for specific therapies. To discover biomarkers of late TNBC recurrence, additional analyses are needed. Citation Format: Judith Passildas-Jahanmohan, Mona Ouled Dhaou, Fabrice Kwiatkowski, Wilfrid Finck, Camille Poirier, Marie-Ange Mouret-Reynier, Xavier Durando, Catherine Abrial, Frederique Penault-Llorca, Nina Radosevic. Pattern and biomarkers of recurrence in 305 triple negative breast cancer patients treated in a French comprehensive cancer center [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 P3-08-45.

Polysaccharides of Huaier Suppress Metastasis and Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Inducing Autophagy to Degrade Snail

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and lacks targeted therapies. Trametes robiniophila Murr (Huaier) has been widely used in clinical anti-cancer treatments in China. Previously, we found that Huaier can effectively improve 5-year overall survival and disease free survival in stage III TNBC patients. The Polysaccharides of Huaier (PS-T) were identified as the major components of Huaier. Further study will be needed to elucidate the efficacy of PS-T as potential anti-tumor adjuvant strategy in TNBC. Methods: Transwell assay, scratch assay and mice lung metastasis model were used to observe cell invasion and migration in vitro and in vivo. Western blotting was used to determine the expression of epithelial-mesenchymal transition (EMT) and autophagy related proteins. Immunofluorescence analysis was used to detect autophagosome-lysosome fusion and protein expression of LC3 and Snail. A cell line MDA-MB-231-siATG5 in which autophagy key protein ATG5 was stably interfered was constructed. An E-cadherin promoter reporter gene plasmid and a Snail overexpression plasmid were sequentially introduced into MDA-MB-231 cells. Immunohistochemistry and Database analysis online(the Human Protein Atlas, UALCAN, Kaplan-Meier plotter) were used to explore the distinct prognostic and potential therapeutic value of Snail in breast cancer patients. Findings: PS-T inhibited cell invasion and migration in vitro and in vivo, and reversed the EMT while induced autophagy. Utilization of autophagy inhibitor LY294002 or knockdown of ATG5 by lentivirus infection suppressed the inhibitory effects of PS-T. Snail, a key transcription factor that controls EMT initiation was found to be degraded by PS-T induced autophagy. Overexpression of Snail reversed the inhibitory effects of PS-T. IHC for breast cancer tissues of 33 cases of TNBC patients and Database analysis online verified that high expression of Snail was associated with poor prognostics. Interpretation: We demonstrate that PS-T can inhibit EMT in breast cancer cell by the induction of autophagy to degrade Snail protein which contributes to both the EMT and metastasis. This study provides a new insight into the mechanism by which PS-T reduce the recurrence of TNBC and new ideas for comprehensive treatment strategies for TNBC patients. Funding Statement: National Natural Science foundation of China (No. 81802664). Declaration of Interests: All authors declare no competing interest. Ethics Approval Statement: Animals were treated according to protocols established by the ethics committee of Army Medical University and the experiments in vivo were conducted according to the approved guidelines and approved by the ethics committee of Army Medical University (AMUWEC2019199).

Deregulating the CYP2C19/Epoxy-Eicosatrienoic Acid-Associated FABP4/FABP5 Signaling Network as a Therapeutic Approach for Metastatic Triple-Negative Breast Cancer.

Recurrence and metastasis are the main causes of triple-negative breast cancer (TNBC) mortality. On the basis of our clinical cohorts and integrative omics analyses, we hypothesized that understanding the interplay between fatty acid binding protein (FABP) and epoxy-eicosatrienoic acid (EET) driven metastatic progression can uncover a new opportunity for TNBC intervention. In this study, the biological relevance of increased protein expression of CYP2C19, FABP4, and FABP5 in TNBC tumors and in the TNBC cell line (MDA-MB-231), as well as its highly metastatic lung seeking variant (LM6) were delineated from publicly available datasets, shRNA-mediated knockdown, EET supplementation, cancer and stromal cell co-cultures, and an orthotopic and resection xenograft tumor mouse model. We found that the high expression levels of CYP2C19 and FABP4 and FABP5 are critical in TNBC metastatic transformation and stromal cell interactions. Furthermore, EET-associated nuclear translocation of FABP4 and FABP5 and nuclear accumulation of SREBP-2 or PPAR-γ influence TNBC cell proliferation, migratory transformation, and distal metastasis priming. Most notably, we uncovered novel bioefficacy and modes of action of the anticancer drug doxorubicin and a phytogalactolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG), which effectively attenuated TNBC recurrence and lung metastasis through deregulating the FABP/EET dynamics and levels. This study, therefore, introduces a novel approach to combating TNBC by targeting the FABP/EET/CYP-associated metastatic signaling network.

Acid-labile polysaccharide prodrug via lapatinib-sensitizing effect substantially prevented metastasis and postoperative recurrence of triple-negative breast cancer.

Surgical resection and chemotherapy are routinely performed for triple-negative breast cancer (TNBC) because it is insensitive to endocrine therapy and molecular targeted therapy. Here, the optimal surface charge (-28 mV) and particle size (51 nm) enabled the acid-labile hyaluronic acid pullulan prodrug (HPP)-doxorubicin (Dox)/lapatinib (Lap) conjugate to circulate in the blood for a lengthy period of time and enhance the electron paramagnetic resonance effect, while the targeted molecule hyaluronic acid accelerated CD44 receptor-mediated 4T1 cell internalization. The inefficient anti-proliferation capability of Lap increased more than 10-fold after sensitization of Dox to metastatic 4T1 cells, while cellular uptake significantly increased, and cell viability dramatically decreased to nearly 20% of the free Dox group. Furthermore, HPP-Dox/Lap more effectively inhibited lateral mobility, vertical migration, and invasion ability of 4T1 cells. The ex vivo biodistribution of representative Dox indicated that Lap obviously facilitated the intratumoral infiltration and accumulation. The in vivo research revealed that there were overwhelming advantages in using HPP-Dox/Lap to inhibit tumor growth, progression, and lung metastasis even at a low dosage (1 mg kg-1), and it decreased postoperative recurrence and pulmonary metastatic nodules. Because of the excellent biosafety and visible therapeutic effect on the 4T1 metastasis and recurrence model, there is great potential value for HPP-Dox/Lap to be used to treat metastatic TNBC.

Cluster of differentiation 8 and programmed cell death ligand 1 expression in triple-negative breast cancer combined with autosomal dominant polycystic kidney disease and tuberous sclerosis complex: a case report

BackgroundAutosomal dominant polycystic kidney disease is defined as an inherited disorder characterized by renal cyst formation due to mutations in the PKD1 or PKD2 gene, whereas tuberous sclerosis complex is an autosomal dominant neurocutaneous syndrome caused by mutation or deletion of the TSC2 gene. A TSC2/PKD1 contiguous gene syndrome, which is caused by a chromosomal mutation that disrupts both the TSC2 and PKD1 genes, has been identified in patients with tuberous sclerosis complex and severe early-onset autosomal dominant polycystic kidney disease. The tumor tissue of patients with breast cancer with contiguous gene syndrome has a high mutation burden and produces several neoantigens. A diffuse positive immunohistochemistry staining for cluster of differentiation 8+ in the T cells of breast cancer tissue is consistent with neoantigen production due to high mutation burden.Case presentationA 61-year-old Japanese woman who had been undergoing dialysis for 23 years because of end-stage renal failure secondary to autosomal dominant polycystic kidney disease was diagnosed as having triple-negative breast cancer and underwent mastectomy in 2015. She had a history of epilepsy and skin hamartoma. Her grandmother, mother, two aunts, four cousins, and one brother were also on dialysis for autosomal dominant polycystic kidney disease. Her brother had epilepsy and a brain nodule. Another brother had a syndrome of kidney failure, intellectual disability, and diabetes mellitus, which seemed to be caused by mutation in the CREBBP gene. Immunohistochemistry of our patient’s breast tissue showed cluster of differentiation 8 and programmed cell death ligand 1 positivity.ConclusionsProgrammed cell death ligand 1 checkpoint therapy may be effective for recurrence of triple-negative breast cancer in a patient with autosomal dominant polycystic kidney disease and tuberous sclerosis complex.

Triple-negative breast cancer: Pretreatment magnetic resonance imaging features and clinicopathological factors associated with recurrence

PurposeWe aimed to investigate the magnetic resonance imaging (MRI) features and clinicopathologic factors with recurrence of triple-negative breast cancer (TNBC). Patients and methodsWe identified 281 patients with 288 surgically confirmed TNBC lesions who underwent pretreatment MRI between 2009 and 2015. The presence of intratumoral high signal on T2-weighted images, high-signal rim on diffusion-weighted images (DWI), and rim enhancement on the dynamic contrast-enhanced MRI and clinicopathological data were collected. Cox proportional analysis was performed. ResultsOf the 288 lesions, 36 (12.5%) recurred after a median follow-up of 18 months (range, 3.6–68.3 months). Rim enhancement (hazard ratio [HR] = 3.15; 95% confidence interval [CI] = 1.01, 9.88; p = .048), and lymphovascular invasion (HR = 2.73, 95% CI = 1.20, 6.23; p = .016) were independently associated with disease recurrence. While fibroglandular volume, background parenchymal enhancement, intratumoral T2 high signal, and high-signal rim on DWI, were not found to be risk factors for recurrence. ConclusionPretreatment MRI features may help predict a high risk of recurrence in patients with TNBC.

Post-Radiotherapy Inflammation Predicts Recurrence and Mortality in Stage I-III Triple-Negative Breast Cancer

Recent studies suggest that a pro-inflammatory lymphopenic microenvironment associated with radiotherapy (RT) may contribute to recurrence of triple-negative breast cancer (TNBC). Furthermore, adiposity may exacerbate this pro-tumor inflammation. To study how RT affects the immune system in this high-risk cancer, we investigated the systemic inflammatory response to RT in patients with TNBC. We hypothesized that inflammation in a lymphopenic setting within one year after RT correlates with recurrence and mortality in stage I-III TNBC. We also hypothesized that obesity would intensify the relapse risk associated with inflammation. We performed a single-institutional retrospective cohort study of adult women treated with definitive RT for stage I-III TNBC between 1999 and 2012. Patients had a minimum follow-up of 5 years. Clinicopathologic characteristics and complete blood counts were recorded. Neutrophil:lymphocyte ratio (NLR), platelet:lymphocyte ratio (PLR), and monocyte:lymphocyte ratio (MLR) were calculated. Time-to-event analyses for recurrence and mortality were analyzed by Cox proportional-hazards regression. Confounders were adjusted using forward stepwise selection to avoid multicollinearity. Optimal inflammatory marker cutoffs from receiver operating characteristic curves were analyzed by log-rank after stratification on Kaplan-Meier plots. After a median follow-up of 6.9 years after RT, 110 patients with stage I (n=32, 29%), stage II (n=53, 48%) and stage III (n=25, 23%) TNBC were studied. We evaluated early (2 mo) and long term (8-12 mo) effects of RT on peripheral immune markers in relation to TNBC outcomes. Within 2 months of RT, elevated neutrophil count was associated with metastasis (Hazard ratio 1.25, p=0.04). Patients with absolute lymphocyte count (ALC) less than 0.55 K/μL (HR 3.12, p=0.04) or monocyte count above 0.5 K/μL (HR 2.67, p=0.03) had greater TNBC-specific mortality. PLR > 345 predicted reduced overall survival (HR 2.50, p=0.04), and patients with MLR > 0.9 had a greater risk of TNBC-specific mortality (HR 5.72, p=0.001). After adjustment, elevated NLR was associated with TNBC-specific mortality (HR 1.2, p=0.04), and high PLR predicted local recurrence (HR 1.01, p<0.05). Sustained lymphopenia 8-12 mo following RT (ALC < 1.16 K/μL) predicted mortality (HR 3.77, p=0.02). Finally, body mass index was an independent poor prognostic factor in each inflammatory model. Systemic inflammation and lymphopenia after RT may represent novel prognostic biomarkers associated with recurrence, metastasis, and mortality in stage I-III TNBC. Further studies evaluating RT immune effects are needed to validate this finding and develop treatment strategies.

Immunologic responses in triple-negative breast cancer patients in a randomized phase IIb trial of nelipepimut-S plus trastuzumab versus trastuzumab alone to prevent recurrence.

556 Background: Breast cancer (BC) patients (pts) expressing low levels of HER2 by (immunohistochemistry (IHC) 1-2+) are not eligible for trastuzumab (Tz). However, in a randomized phase 2b trial, triple negative BC (TNBC) pts demonstrated a significantly better DFS with nelipepimut-S (NPS) + Tz vs Tz alone. Here, we assess the ex vivo and in vivo immune responses (IR) in both arms. Methods: Disease-free pts (n = 275) with HER2 IHC 1-2+, non-amplified BC who were node positive and/or had TNBC were randomized 1:1 to granulocyte-macrophage-colony stimulating factor (GM-CSF) or NPS+GM-CSF. ±NPS was given every 3 weeks x 6 followed by 4 boosters every 6 months (mo). All pts received Tz concurrently for 1 year per label regimen and were followed for recurrence. IR were evaluated ex vivo by clonal expansion of NPS-specific cytotoxic T lymphocytes (CTL) by dextramer-staining/flow cytometry at time points over 3 years. In vivo IR were assessed by delayed type hypersensitivity (DTH) reactions periodically. Results: The trial enrolled 97 TNBC pts; 60 had 4 timepoints available for analysis (37 NPS + Tz pts; 23 Tz pts). The NPS+Tz group exhibited increases in CTL frequencies vs baseline: 208%, 303%, 379% at 18, 24 and 30 mo, respectively. NPS+Tz pts’ mean CTL frequencies increased from 0.029 ±0.001% at baseline to 0.112±0.026% at 30 mo (p = 0.01) compared to Tz pts who were 0.027 ±0.001% at baseline and 0.057 ±0.016% at 30 mo (p = 0.71). Only 4 NPS+Tz pts recurred as compared to 13 in the Tz arm. While limited by low numbers, recurrent NPS + Tz pt did not mount an IR by ex vivo assessment (range: 0.0 - 0.026%) or by DTH (all measurements: 0 mm), while non-recurrent pts mounted both clonal CTL expansion (range: 0.000- 0.33%) and enhanced DTH (range: 0.0- 80.5mm). Conclusions: NPS+Tz combination is more efficacious in generating time-dependent antigen (NPS)-specific CTL by both ex vivo and in vivo measures vs Tz. Based on these preliminary data, it appears that both ex vivo and in vivo IR to NPS were attenuated in pts with TNBC recurrence. Further analysis of read-outs from these assays to validate the relationship of IRs to clinical effect seen with NPS+Tz in TNBC pts is underway. Clinical trial information: NCT02297698.