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Supplementary data Fig.1 Study design Supplementary data Fig.2 Flow chart of patient receiving FDG-PET and FLT-PET

Purpose: To evaluate the feasibility of early metabolic change assessed by PET in predicting clinical response to chemotherapy and investigate its prognostic value in patients with advanced gastric cancer.Experimental Design: A total of 64 patients with advanced gastric cancer were prospectively enrolled and examined by PET with 18F-fluorodeoxyglucose (FDG) and 18F-fluoro-3′-deoxy-3′-L-fluorothymidine (FLT) at baseline and 14 days after treatment initiation. PET findings were analyzed for the correlation with best clinical response of patients, disease control status, and survival after identifying the threshold of metabolic change percentage by ROC analysis.Results: For FDG-PET, the total uptake value reduction percentage (δ-SUV) of 40% was the cut-off point with the maximum of sensitivity (70%) and specificity (83%) to predict clinical responding and that of prediction for disease control status was 30%, with the highest sensitivity (58%) and specificity (100%). The δ-SUV of FLT-PET played no predictive role for clinical response (AUC = 0.62; P = 0.134) and disease control (AUC = 0.66; P = 0.157). The univariate Cox regression analysis revealed no significant prognostic impact. FDG uptake reduction in liver metastases could predict both clinical response (P = 0.010) and disease control status (P = 0.002) at thresholds of 35% and 15%, respectively. Those with greater FDG uptake reduction in liver lesions had a longer overall survival (P = 0.004).Conclusions: Early metabolic change in FDG-PET might be a predictive marker for response and disease control in advanced gastric cancer. Early FDG uptake change in liver metastases might be a useful prognostic factor and needs further exploration. Clin Cancer Res; 22(7); 1603–10. ©2015 AACR.

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling–liquid chromatography–tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.

Loss of polarity protein Par3 promotes breast cancer tumorigenesis and metastasis. The underlying molecular mechanisms of Par3 down-regulation and related prognostic significance in breast cancer remain unclear. Here, we discovered that Par3 down-regulation was associated with shorter relapse-free survival in Luminal A subtype of breast cancer. Par3 knockdown promoted breast cancer cells migration and invasion. Importantly, we identified that transcription factor Sp1 bound to PARD3 promoter region and induced Par3 expression. Breast cancer patients with low Sp1 showed significantly worse RFS and low expression level of Par3. Par3 over-expression partially reversed Sp1 knockdown induced migration and invasion. Together, decreased Sp1 level mediates Par3 down-regulation, which correlated with poor prognosis of ER + breast cancer patients, via reduced binding with PARD3 promoter.

Background: This study aimed to evaluate tumor heterogeneity of metastatic breast cancer (MBC) and investigate its impact on the efficacy of pyrotinib in patients with HER2-positive MBC. Methods: MBC patients who underwent 18F-FDG PET/CT before pyrotinib treatment were included. Temporal and spatial tumor heterogeneity was evaluated by the discordance between primary and metastatic immunohistochemistry (IHC) results and baseline 18F-FDG uptake heterogeneity (intertumoral and intratumoral heterogeneity indexes: HI-inter and HI-intra), respectively. Progression-free survival (PFS) was estimated by the Kaplan–Meier method and compared by a log-rank test. Results: A total of 572 patients were screened and 51 patients were included. In 36 patients with matched IHC results, 25% of them had HER2 status conversion. Patients with homogenous HER2 positivity had the longest PFS, followed by patients with gained HER2 positivity, while patients with HER2 negative conversion could not benefit from pyrotinib (16.8 vs. 13.7 vs. 3.6 months, p < 0.0001). In terms of spatial heterogeneity, patients with high HI-intra and HI-inter had significantly worse PFS compared to those with low heterogeneity (10.6 vs. 25.3 months, p = 0.023; 11.2 vs. 25.3 months, p = 0.040). Conclusions: Temporal heterogeneity of HER2 status and spatial heterogeneity of 18F-FDG uptake could predict the treatment outcome of pyrotinib in patients with HER2-positive MBC, which provide practically applicable methods to assess tumor heterogeneity and guidance for treatment decisions.

Background The microRNA (miRNA) miR-133a-1 has been identified as a tumor suppressor in breast cancer. However, the underlying mechanisms of miR-133a-1 in breast cancer have not been fully elucidated. This study aimed to explore the targets of miR-133a-1 in breast cancer using an integrated bioinformatics approach. Methods Human SKBR3 breast cancer cells were transfected with miR-133a-1 or a miRNA negative control (miRNA-NC) for 48 hours. The RNA-seq sequencing technique was performed to identify the differential expression of genes induced by miR-133a-1 overexpression. Functional enrichment analysis was conducted to determine the target genes and pathways involved in breast cancer. Results Breast cancer patients with high levels of miR-133a-1 expression commonly showed longer overall survival compared to patients with a low level of miR-133a-1 expression. Using Cuffdiff, we identified 1,216 differentially expressed genes induced by miR-133a-1 overexpression, including 653 upregulated and 563 downregulated genes. MOCS3 was the most upregulated gene and KRT14 was the most downregulated gene. The top 10 pathways related to the differentially expressed genes were identified through Gene Ontology (GO) enrichment analysis. Sex-determining region Y-box 9 (SOX9) demonstrated the highest semantic similarities among the differentially expressed genes. Since SOX9 and CD44 were hub nodes in the protein-protein interaction network, the SOX9 gene may be a target of miR-133a-1 in breast cancer. Conclusions This report provides useful insights for understanding the underlying mechanisms in the pathogenesis of breast cancer.

Immune checkpoint inhibitors (ICIs) have revolutionized the therapeutic landscape of cancer and have been widely approved for use in the treatment of diverse solid tumors. Targeted therapy has been an essential part of cancer treatment for decades, and in most cases, a special drug target is required. Numerous studies have confirmed the synergistic effect of combining ICIs with targeted therapy. For example, triple therapy of PD-L1 inhibitor atezolizumab plus BRAF inhibitor vemurafenib and MEK inhibitor cobimetinib has been approved as the first-line treatment in advanced melanoma patients with BRAFV600 mutations. However, not all combinations of ICIs and targeted therapy work. Combining ICIs with EGFR inhibitors in non-small-cell lung cancer (NSCLC) with EGFR mutations only triggered toxicities and did not improve efficacy. Therefore, the efficacies of combinations of ICIs and different targeted agents are distinct. This review firstly and comprehensively covered the current status of studies on the combination of ICIs mainly referring to PD-1 and PD-L1 inhibitors and targeted drugs, including angiogenesis inhibitors, EGFR/HER2 inhibitors, PARP inhibitors and MAPK/ERK signaling pathway inhibitors, in the treatment of solid tumors. We discussed the underlying mechanisms, clinical efficacies, side effects, and potential predictive biomarkers to give an integrated view of the combination strategy and provide perspectives for future directions in solid tumors.

Heterogeneity represents a pivotal factor in the therapeutic failure of triple-negative breast cancer (TNBC). In this study, we retrospectively collected and analysed clinical and pathological data from 258 patients diagnosed with TNBC at the Fudan University Cancer Hospital. Our findings show that low ARID1A expression is an independent prognostic indicator for poor overall survival (OS) and recurrence-free survival (RFS) in TNBC patients. Mechanistically, both nuclear and cytoplasmic protein analyses and immunofluorescent localisation assays confirm that ARID1A recruits the Hippo pathway effector YAP into the nucleus in human triple-negative breast cancer cells. Subsequently, we designed a YAP truncator plasmid and confirmed through co-immunoprecipitation that ARID1A can competitively bind to the WW domain of YAP, forming an ARID1A/YAP complex. Moreover, the downregulation of ARID1A promoted migration and invasion in both human triple-negative breast cancer cells and xenograft models through the Hippo/YAP signalling axis. Collectively, these findings demonstrate that ARID1A orchestrates the molecular network of YAP/EMT pathways to affect the heterogeneity in TNBC.

Background This study investigated the incidence, radiographic patterns, and relevance to clinical outcome of everolimus-related pneumonitis (ERP) in patients with metastatic breast cancer (MBC). Materials and Methods Data of patients with MBC treated with everolimus who had baseline and at least one follow-up chest computed tomography (CT) were obtained from a medical electronic database system. An independent review of the CT scans of these patients was conducted by two radiologists (NCT 03730428). Log-rank and Cox proportional hazard regression analyses were used for time-to-event analyses. Results ERP was radiographically detected in 45 of 86 patients (52.3%). In more than 80% of these patients, ERP occurred during the first 4 months of everolimus treatment. Only 14 of the 45 patients with ERP were symptomatic (31.1%). Symptoms included cough, fever, and shortness of breath. Bilateral and lower distribution of the pneumonitis was most common. In most of the cases, ground-glass opacities and reticular opacities were noticed. Elderly patients were more likely to develop ERP. Patients with ERP had significantly longer progression-free survival (PFS; 6.8 vs. 4.1 months, p = .024) and overall survival (OS; 42.8 vs. 21.3 months, p = .016). ERP was a predictor of OS improvement confirmed by multivariate Cox analysis (hazard ratio, 0.49; 95% confidence interval, 0.25–0.97; p = .040). Conclusions ERP was noted in half of the patients with MBC treated with everolimus. Our data suggested that ERP was associated with improved prognosis and may be used as a biomarker for the efficacy of everolimus in MBC. Close monitoring, prompt diagnosis, and proper treatment for ERP are essential to maintain the quality of life of patients and achieve maximum treatment benefits. Implications for Practice Everolimus-related pneumonitis (ERP) is one of the most worrying drug adverse events, especially in Asian patients. However, little has been known about the clinical and radiographic details of ERP in patients with metastatic breast cancers (MBCs) treated with everolimus. The present study investigated the clinical characteristics, radiographic patterns, and its correlation with treatment outcome in patients with MBC. ERP was identified in more than half of patients with MBC during everolimus therapy and was associated with improved outcome. Close monitoring and prompt diagnosis and appropriate treatment for ERP are critical for the preservation of patients' quality of life and achievement of maximal treatment benefits.

Background Early integration of palliative care (PC) for patients with advanced cancer has been recommended to improve quality of care. This study aims to describe prevalence, temporal trend and predictors of PC use in metastatic breast cancer (mBCa) patients receiving critical care therapies (CCT; included invasive mechanic ventilation, percutaneous endoscopic gastrostomy tube, total parenteral nutrition, tracheostomy and dialysis). Methods The National Inpatient Sample was queried for mBCa patients receiving CCT between 2005 and 2014. Annual percent changes (APC) were calculated for PC prevalence in the overall cohort and subgroups. Multivariable logistic analysis was used to explore predictors of PC use. Results Of 5833 mBCa patients receiving CCT, 880 (15.09%) received PC. Rate of PC use increased significantly from 2.53% in 2005 to 25.96% in 2014 (APC: 35.75%; p, Highlights • Overall, 15.09% of mBCa patients with CCT receive inpatient PC. • Rate of inpatient PC use in mBCa patients receiving CCT increases significantly from 2005 to 2014. • This study highlights the underutilized PC in mBCa patients receiving CCT. • Future efforts to illustrate disparities in PC use are needed to improve quality of care.

To explore the role of DNA repair gene ERCC1 in breast cancer metastasis and its signaling pathway.Through prospective cohort studies and cell tests, Western Blot and IHC were used to detect the expression of DNA repair gene ERCC1 and NF-κB signaling pathways related proteins, scratch test and Transwell to detect the ability of migration and invasion. To explore the mechanism of ERCC1 affecting breast cancer metastasis. Population results: There was no significant difference in clinicopathological features between the two groups (P > 0.05). ERCC1 has a positive correlation with breast cancer metastasis(r = 0.543, P Χ2 = 17.571, P P P r = 0.481, P r = -0.203, P r = -0.275, P r = 0.386, P r = 0.476, P r = 0.281, P P P P P P P

Fulvestrant 500 mg is standard of care for endocrine therapy‐naive or pretreated women with hormone receptor‐positive (HR+) metastatic breast cancer (MBC). This study was conducted to explore the potential factors and duration of last endocrine therapy as predictors for the efficacy of fulvestrant 500 mg on Chinese patients in real‐world practice. Two hundred and fifty‐two MBC patients who were treated with fulvestrant 500 mg consecutively between January 2011 and December 2015 in our institute were included in this study. Efficacy outcomes included progression‐free survival (PFS), overall survival (OS), and clinical benefit rate (CBR). The optimal cut‐off value for duration of last endocrine therapy was determined by survival ROC analysis. Adverse events were graded according to NCI‐CTC AE 4.0. Fulvestrant 500 mg demonstrated a median PFS of 5.8 months (95%CI 4.6‐6.9), and a median OS of 35.9 months (95%CI 30.2‐41.4). CBR was 41.3% (95%CI 35‐47). Liver metastasis, bone alone metastasis, lines of endocrine therapy for MBC, and sensitivity to last endocrine therapy were statistically significant in the Cox multivariate analysis (P values of 0.022, 0.02, 0.03, and 0.038, respectively). The optimal cut‐off values for duration of last endocrine therapy to predict the efficacy of fulvestrant 500 mg were 25.08 months for adjuvant endocrine therapy and 5.17 months for first‐line endocrine therapy, which showed no difference in prediction power with ABC clinical definition. Patients with prior adjuvant endocrine therapy ≥25.08 months or first‐line therapy≥5.17 months reached a longer PFS of fulvestrant (p = 0.04). Six patients discontinued the treatment due to intolerable adverse events. Patients with the duration of prior endocrine therapy longer than optimal cut‐off points indicate better PFS of fulvestrant. Liver metastasis, bone alone metastasis, line of fulvestrant, and sensitivity to last endocrine therapy were also predictors for response of fulvestrant. ClinicalTrials.gov Identifier: NCT03708432., This study provided first‐hand data regarding the efficacy and safety profile of fulvestrant 500 mg in Chinese patients with HR+/HER2‐ MBC. We successively collected all patients treated with fulvestrant within a certain period of time to delineate the pattern of real‐world practice. The study identified potential determinants that were significantly associated with PFS of fulvestrant 500 mg.

BACKGROUND Platinum-based chemotherapy (PBCT) has gained an important position as a first-line treatment for metastatic triple-negative breast cancer (mTNBC). We assessed whether maintenance chemotherapy maintenance was superior to observation after first-line PBCT in patients with mTNBC. METHODS A total of 265 patients with mTNBC who had exhibited non-PD after 4-6 cycles of firstline PBCT at the Fudan University Shanghai Cancer Center from January 2008 to April 2019 were retrospectively analyzed. 107 patients who did not receive additional treatment were defined as the control observation group, and the remaining 158 patients who continued to receive maintenance therapy were defined as the maintenance treatment group. RESULTS The median progression-free survival (PFS) time in the maintenance group was 9.63 months, which was significantly longer than the PFS time of 7.47 months in the observation group (HR 0.49, 95% CI: 0.37-0.67, P

Purpose Malic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis. Its function in breast cancer remains to be fully clarified. The aim of this work was to investigate the prognostic value of ME1 and its possible mechanism in breast cancer. Methods We evaluated ME1 expression in 220 early breast cancer patients with tissue microarray-based immunohistochemistry and explored the relationships between ME1 expression and clinicopathological features. Survival analyses were further performed to determine its prognostic value. The public database was used to confirm tissue microarray results. Further, cell proliferation, migration, invasion ability and reactive oxygen species (ROS) were examined in breast cancer cells. Results In breast cancer tissues, high ME1 expression was significantly associated with larger tumor size, higher incidence of lymph node metastasis and higher incidence of lymph-vascular invasion. High ME1 expression significantly correlated with worse recurrence-free survival (RFS), and was an independent prognostic factor for RFS, which was confirmed by mRNA results in the public database. In vitro, upregulation of ME1 by transfecting MCF-7 cells with virus vector remarkably enhanced viability, motility and epithelial-mesenchymal transition (EMT) and decreased ROS levels, whereas knockdown in MDA-MB-468 cells produced totally opposite effects as expected. When pretreated with oxidizing agent, MCF-7 cells overexpressing ME1 lost its motility, whereas MDA-MB-468 cells with knockdown of ME1 restored its motility when pretreated with antioxidant. Conclusion To our knowledge, these clinical and experiment works first suggested that ME1 may be a novel biomarker and potential therapeutic target for breast cancer metastasis, and its biological effect is mainly controlled by manipulating ROS.

Background Although impact of surgery on survival for de novo metastatic breast cancer (MBC) patients has been debated for decades, it remains controversial. We sought to evaluate whether surgery of primary site (SPS) and surgery of metastatic site (SMS) provide survival benefits in a large cohort. Methods A retrospective population-based cohort study was conducted by using data from the 2010-2015 Surveillance, Epidemiology, and End Results (SEER) program. Descriptive statistical analysis was used to compare demographic, tumorous and therapeutic characteristics. Median survival was estimated by Kaplan-Meier method. Cox regression models were used to assess risk of breast cancer related death and all-cause death between de novo MBC patients who underwent SPS or SMS and who did not. To minimize selection bias, we conducted additional analysis in patients who were recommended to SPS. Nomograms were constructed based on the Cox regression model to predict 3-year overall survival (OS) and breast cancer specific survival (BCSS) for patients who were recommended to SPS. The nomograms were further internal validated and assessed by calibration curves. Results There were 12443 patients eligible for the study, of whom 4482 patients were recommended to SPS and 3869 patients ultimately performed SPS. Median BCSS and OS of patients were longer in SPS group than non-SPS group, both in the entire population (49.00 vs. 27.00months, P Citation Format: Zhe Feng, Xinyu Chen, Jian Zhang, Zhonghua Tao, Xichun Hu. Surgical intervention improves survival in patients with de novo metastatic breast cancer: A population-based analysis [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 P1-20-11.

Background and purpose In non-small-cell lung carcinoma (NSCLC) patients, the L858R/T790M mutation of the epithelial growth factor receptor (EGFR) is a major cause of acquired resistance to EGFR-TKIs treatment that limits their therapeutic efficacy. Identification of drugs that can preferentially kill the NSCLC harbouring L858R/T790M mutation is therefore critical. Here, we have evaluated the effects of ursolic acid, an active component isolated from herbal sources, on erlotinib-resistant H1975 cells that harbour the L858R/T790M mutation. Experimental approach Gene expression omnibus (GEO) profiles analyses was applied to detect differentially expressed genes in NSCLC cells harbouring EGFR mutation. AnnexinV-FITC/PI, TUNEL staining, MTT, wound healing, RT-PCR, qRT-PCR, western blots, immunostaining, dual-luciferase reporters and ChIP-PCR were utilized to investigate the effects of ursolic acid in vitro and in vivo. Key results The cancer/testis antigen family 45 member A2 (CT45A2) was highly expressed in H1975 cells. Ectopic expression of CT45A2 in H1975 cells increased cell proliferation and motility in vitro. Silencing the CT45A2 expression strongly attenuated H1975 cells motility and growth. The anti-cancer effect of ursolic acid was critically dependent on CT45A2 expression in H1975 cells. Ursolic acid suppressed CT45A2 gene transcription mediated by transcriptional factor TCF4 and β-catenin signalling. Conclusions and implications CT45A2 is a novel oncogene for NSCLC with an EGFR T790 mutation. Ursolic acid induced apoptosis and inhibited proliferation of H1975 cells by negatively regulating the β-catenin/TCF4/CT45A2 signalling pathway. Therefore, ursolic acid may be a potential candidate treatment for NSCLC harbouring the EGFR-L858R/T790M mutation.

Purpose Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis. Methods Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis. Results MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells. After ingesting exosomes enriched with MMP-1, cells secrete more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both up-regulated. Clinically, the exosomal MMP-1 can be detected from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of exosomal MMP-1 is also higher than that in patients with single lesion. Conclusion MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.

SummaryPurpose. This trial aimed to evaluate the efficacy and safety of mitoxantrone hydrochloride liposome injection (Lipo-MIT) in advanced breast cancer (ABC). Methods. In this randomized, open-label, active-controlled, single-center, phase II clinical trial, eligible patients were randomized in a ratio of 1:1 to receive Lipo-MIT or mitoxantrone hydrochloride injection (MIT) intravenously. The primary endpoint was objective response rate (ORR). The secondary endpoints were disease control rate (DCR), progression-free survival (PFS), and safety outcomes. Results. Sixty patients were randomized to receive Lipo-MIT or MIT. The ORR was 13.3% (95% confidence interval (CI): 3.8–30.7%) for Lipo-MIT and 6.7% (95% CI: 0.8–22.1%) for MIT. The DCR was 50% (95% CI: 31.3–68.7%) with Lipo-MIT vs. 30% (95% CI: 14.7–49.4%) with MIT. The median PFS was 1.92 months (95% CI: 1.75–3.61) for Lipo-MIT and 1.85 months (95% CI: 1.75–2.02) for MIT. The most common toxicity was myelosuppression. Lipo-MIT resulted in an incidence of 86.7% of leukopenia and 80.0% of neutropenia, which was marginally superior to MIT (96.7% and 96.7%, respectively). Lipo-MIT showed a lower incidence of cardiovascular events (13.3% vs. 20.0%) and increased cardiac troponin T (3.3% vs. 36.7%); but higher incidence of anemia (76.7% vs. 46.7%), skin hyperpigmentation (66.7% vs. 3.3%), and fever (23.3% vs. 10.0%) than MIT. Conclusions The clinical benefit parameters of Lipo-MIT and MIT were comparable. Lipo-MIT provided a different toxicity profile, which might be associated with the altered distribution of the drug. Additional study is needed to elucidate the potential benefit of Lipo-MIT in ABC. Clinical trial registration. This study is registered with ClinicalTrials.gov (No. NCT02596373) on Nov 4, 2015.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis.Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. MMP-1 level was detected using mass spectrometry and western blot. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis.MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells, including MDA-MB-231, MDA-MB-468 and BT549. After ingesting exosomes enriched with MMP-1, cells secret more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The lung colonization model shows that the expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both upregulated. Clinically, the enrichment of MMP-1 can be detected in exosomes extracted from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of MMP-1 in exosomes is also higher than that in patients with single lesion.MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.

Background: Breast cancer is a malignancy with no clearly identified prognostic factors for diagnosis. Studies have preliminarily found that lncRNAs are related to breast cancer metastasis, however, the clinical predictive significance of lncRNAs is still elusive.In this study, we evaluated the diagnostic value of long non-coding RNA (lncRNA UCA1,CCAT2, ANCR) on postoperative metastasis of breast cancer as well as the possible mechanism involving the EMT. Methods: We investigated lncRNA ANCR, UCA1,CCAT2 that associated with breast cancer metastasis risk in a population-based nested case-control study. Metastasis cases were identified by clinical diagnostic criteria in approximately 103 cases in the Cancer Institute of Southwest Medical University during 2013-2020. At the same time, the control group (metastasis-free) was selected according to the 1:1 pairing principle in this cohort (n=103, the matching condition was age±3 years, the operation time within the same month, and the treatment plan both are modifed radical mastectomy) The mRNA of lncRNA( UCA1,CCAT2, ANCR) expression was determined by Real-time PCR. The expression of E-cadherin, N-cadherin, and vimentin proteins was detected by Western blot. The migration and invasion of transfected cells were determined by the Transwell assay.Results: lncRNA ANCR, UCA1, CCAT2 was significantly up-regulated in breast cancer cells and postoperative metastasis of breast cancer.CCAT2 (OR=1.024, 95% CI: 1.010, 1.039), UCA1(OR=1.025, 95% CI: 1.011, 1.039),ANCR(OR=1.055, 95% CI:1.001, 1.111)was the risk factor for postoperative metastasis of breast cancer. Furthermore , we used the ROC curve to detect the optimal critical values of CCAT2, UCA1, ANCR , the risk of metastasis in the CCAT2 high expression group was 2.297 times that of the low expression group (OR=2.297, 95% CI:1.427 ~ 3.695, P< 0.05). The risk of metastasis in the UCA1 high expression group was 2.032 times that of the low expression group (OR=2.032, 95% CI 1.282 ~ 3.218, PConclusions: Our data suggested that lncRNA CCAT2 , UCA1,ANCR was a novel molecule involved in postoperative metastasis of breast cancer, which has predictive value in patients with breast cancer metastasis.

Objectives Carcinosarcoma of the breast is a rare disease. Its clinicopathological features and prognosis are not well defined. The aim of this study was to compare the clinicopathological features and clinical outcome between breast carcinosarcoma and breast invasive ductal carcinoma (IDC). Materials and methods Patients with breast carcinosarcoma and breast IDC were identified through the Surveillance, Epidemiology, and End Results (SEER) database from 2010 to 2015. Then a comparison was conducted between these two groups. Propensity score matching (PSM) was performed to balance the effects of baseline clinicopathological differences. The Cox proportional hazard model was used to identify potential prognostic factors of breast carcinosarcoma. Results In total, we identified 63 patients with breast carcinosarcoma and 200,596 cases with breast IDC. Comparing with IDC, breast carcinosarcoma was significantly correlated with higher grading, higher staging, larger tumor size, lower lymph node involvement, and a higher proportion of triple negative breast cancer (TNBC), suggesting a significantly worse clinical outcome. After adjusting for the uneven clinicopathological variables with PSM, significant differences were still observed between these two histology types. Subgroup analysis further showed that carcinosarcoma-TNBC has an inferior clinical outcome compared with IDC-TNBC. Finally, we identified independent prognostic factors, namely, stage, tumor size, and distant metastasis. Conclusion It is concluded that breast carcinosarcoma has distinct clinicopathological features and a significantly worse clinical outcome than common IDC., Highlights • Breast carcinosarcoma correlates with a higher proportion of triple negative breast cancer than invasive ductal carcinoma. • Triple negative breast carcinosarcoma correlates with worse prognosis than triple negative invasive ductal carcinoma. • Stage, tumor size, and distant metastasis were independent prognostic factors for breast carcinosarcoma.

In this study, the authors have successfully prepared the polyethylene glycol (PEG)‐coated zinc oxide nanoparticles (ZNPs) and studied its effect in pancreatic cancer cells. The authors have observed a nanosized particle with spherical shape. In this study, the authors have demonstrated the cytotoxic effect of ZNP and PZNP in PANC1 cells. To be specific, PZNP was more cytotoxic compared to that of ZNP in PANC1 cancer cells. The authors have further showed that apoptosis is the main mode of cytotoxic activity. It is worth noting that PEGylation of ZNP did not decrease the cell killing activity of zinc particles, whereas it further increases its anticancer effect in the pancreatic cancer cells. The authors have observed a significant upregulation of proapoptotic BAX while expression of antiapoptotic Bcl‐2 was significantly downregulated indicating the potent anticancer effect of zinc nanoparticles. Overall, PEGylation of ZNP could be an effective strategy to improve the stability, while at the same time, its anticancer activity could be enhanced for better therapeutic response.

Background: Malic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis, playing a part in the Warburg effect. Recently, several researches have revealed its crucial role in cancer metastasis. KM-plotter, an online analysis tool, showed high ME1 mRNA level was related to poorer RFS and OS. Herein, we explore the prognostic effect of ME1 on invasive breast cancer in Chinese patients and its effect on metastasis in vitro. Methods: 220 patients with early breast cancer were included in this study. ME1 expression was evaluated semi-quantitatively with tissue microarray-based immunohistochemistry. The relationships between ME1 expression level and clinicopathological features were explored. Survival analyses were carried out by Kaplan-Meier test and COX proportional hazard model. MCF-7 and MDA-MB-468 cell lines were then used for in vitro cell migration and invasion assays. Results: High expression of ME1 was observed in 51.5% patients. The median follow-up period was 28.9 months (range 0.5-34.0). In correlation analyses, compared with ME1-low cases, ME1-high cases were significantly associated with larger tumor size (P=0.036), positive lymph nodes (P Conclusions: ME1 enhances migration and invasion of breast cancer cell lines and may be involved in early development of breast cancer metastasis. Thus, ME1 is a promising prognostic indicator and a potential therapeutic target. Citation Format: Liu C, Tao Z, Hu X. Malic enzyme 1 is a potential metastasis-related biomarker of breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-45.

Background Platinum-based regimens are the mainstay treatments for advanced triple-negative breast cancer (TNBC). Preclinical studies have shown that the histone deacetylase (HDAC) inhibitor chidamide induced antitumor effects in TNBC, and chidamide plus chemotherapy was shown to be tolerable in several malignancies. This study sought to investigate the efficacy and safety of a combination treatment of chidamide and cisplatin in metastatic TNBC patients. Methods In this phase II, single-arm study, women with metastatic TNBC were administered chidamide (20 mg twice weekly for 2 weeks on a 21-day cycle) and cisplatin (75 mg/m2 on a 21-day cycle). The primary endpoint was the objective response rate (ORR) by RECIST 1.1. The severity of adverse events was measured by the CTCAE 4.03. Results Sixteen patients were enrolled in this study. Of these, 15 were available for evaluation. In these 15 patients, confirmed objective responses were seen in 4 patients [26.67%, 95% confidence interval (CI): 10.9%, 51.95%]. The ORRs did not meet the predefined criteria (of a response by at least 5 of the 15 patients); thus, the study remained at stage I. The median progression-free survival (PFS) was 9.8 weeks; 4 patients had a PFS of >25 weeks. In relation to the treatment-related AEs ≥ grade 3, >2 patients had neutropenia (33%), thrombocytopenia (20%), leucopenia (20%), and vomiting (20%). Conclusions The addition of chidamide did not improve the efficacy of cisplatin in the first-line treatment against advanced TNBC; thus, the phase II clinical trial did not progress any further. Our study appears to be the first to investigate the HDAC inhibitor in TNBC patients and showed disappointing results, which should inform future studies. Future research on cisplatin-based combination treatments for TNBC should consider selecting patients based on predictive biomarkers to increase the clinical benefits.

Background Sesquiterpene lactones have gained tremendous attention owing to their potent anticancer properties. The main focus of this study was to evaluate the anticancer effects of a naturally occurring sesquiterpene lactone, santonin, against human breast cancer SK-BR-3 cells. Material/Methods Cell counting kit 8 assay was used for the determination of cell viability. Apoptosis was detected by DAPI (4′,6-diamidino-2-phenylindole) and annexin V/propidium iodide (IP) staining. Flow cytometry was used for cell cycle analysis and western blotting was used for the estimation of protein expression. Results Results showed that santonin exerts significant anti-proliferative effects on the SK-BR-3 breast cancer cells in a concentration dependent manner. Santonin showed an IC50 of 16 μM against SK-BR-3 cells. DAPI staining showed that santonin caused DNA fragmentation in the SK-BR-3 cells, which is indicative of apoptosis. Annexin V/PI staining showed that apoptotic cell percentage increased up to 34.32% at 32 μM concentration of santonin. Santonin also caused an increase in the expression of Bax, caspase-3, and caspase-9, and a decrease in the expression of Bcl-2. Santonin also caused the arrest of the SK-BR-3 cells at the G2/M phase of the cell cycle and suppressed the expression of cyclin A and B1. Finally, santonin could also block the Raf/MEK/ERK pathway in breast cancer cells. Conclusions The findings of this study suggest the potential for the naturally occurring sesquiterpene lactone santonin in breast cancer treatment and also suggest that it could be developed as a promising anticancer agent.

This study was performed to investigate the effects of lanostane against human breast cancer cells with emphasis on its potential to inhibit cancer cell growth and metastasis along with understanding the underlying molecular mechanism mediating the effects.The SK-BR-3 normal breast line and the MB-157 breast cancer cell line were used in this study. MTT of cell growth was used to determine the viability of cells under lanostane treatment. Colony formation assay was used to analyze the clone forming capability of cancer cells when treated with lanostane. DAPI and acridine orange (AO)/ethidium bromide (EB) staining assays were performed for assessing the apoptic cell death. The level of cellular apoptosis was further examined using flow cytometry. Wound healing and transwell assays were performed to determine the migration and invasion of cancer cells. Western blotting was used for determining the concentration of proteins of interest.The lanostane treatment of cancer cells resulted in loss of cell viability. The IC50 value was 15µM and the inhibitory effects were dose-dependent. However, the inhibition of cell proliferation in normal breast cells was comparatively lower. The antiproliferative effects of lanostane were modulated through Bax/Bcl-2 pathway inducing apoptosis of cancer cells. Furthermore, the lanostane rendered cancer cells less motile and reduced their metastasis remarkably. The inhibition of cell metastasis was modulated through Rho-associated kinases (ROCK) signaling pathway which is involved in metastasis of breast cancer to bone tissues. Hence, the results suggested that lanostane inhibited the breast cancer metastasis to bone.The results of the present study are suggestive of anticancer effects of lanostene triterpene which exerted its effects by inhibiting cell proliferation and metastasis of breast cancer cells mediated through inactivation of Rho-associated kinase signaling. The study holds promise to provide a lead for exploring the secondary metabolite-based anticancer approach against various human malignancies.

Purpose:Triple-negative breast cancer is characterized by fast progression with high possible for metastasis and poor survival. Dysfunction of microRNAs plays an important role in the initiation and progression of cancer. Our previous microRNA-seq data indicated the downregulation of miR-331-3p in triple-negative breast cancer tissues compared with that of the noncancer tissues. However, the function of miR-331-3p in triple-negative breast cancer remains largely unknown. Herein, the involvement of miR-331-3p in triple-negative breast cancer was investigated and the therapeutic potential of miR-331-3p was also explored.Methods:Real-time quantitative polymerase chain reaction was performed to detect the expression of miR-331-3p in triple-negative breast cancer tissues and cell lines. The cell proliferation was determined by the cell counting kit-8 assay. Apoptosis of triple-negative breast cancer cells was examined by annexin V/propidium iodide staining. miRDB database was used to predict the potential targets of miR-331-3p. Western blot was performed to examine the expression of the target protein.Results:miR-331-3p was significantly downregulated in triple-negative breast cancer tissues and cell line. Lower miR-331-3p expression was significantly correlated with the tumor size, TNM stage, and lymph node metastasis of patients with triple-negative breast cancer. Functional experiments showed that the overexpression of miR-331-3p inhibited the proliferation and increased apoptosis of triple-negative breast cancer cells. Neuropilin-2 was identified as a target of miR-331-3p, which harbored binding site of miR-331-3p in its 3′-untranslated region. Overexpression of miR-331-3p decreased the messenger RNA and protein levels of neuropilin-2 in triple-negative breast cancer cells. Restoration of neuropilin-2 partially reversed the inhibitory effects of miR-331-3p on the proliferation of triple-negative breast cancer cells.Conclusions:Our results demonstrated the novel function of miR-331-3p/neuropilin-2 signaling in regulating the malignant behaviors of triple-negative breast cancer cells, which suggested miR-331-3p as a potential target for the treatment of triple-negative breast cancer.

Ibrutinib, an FDA approved, orally administered BTK inhibitor, has demonstrated high response rates to diffuse large B-cell lymphoma (DLBCL), however, complete responses are infrequent and acquired resistance to BTK inhibition can emerge. The present study investigated the role of the platelet-derived growth factor D (PDGFD) gene and the ibrutinib resistance of DLBCL in relation to epidermal growth factor receptor (EGFR). Bioinformatics was used to screen and analyze differentially expressed genes (DEGs) in complete response (CR), partial response (PR) and stable disease (SD) in DLBCL treatment with ibrutinib, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to analyze enriched the signaling pathways increasing DEGs. The Search Tool for Interactions of Chemicals database was used to analyze the target genes of ibrutinib. An interaction network of DEGs, disease-related genes and ibrutinib was constructed. The expression of PDGFD in tissues that were resistant or susceptible to DLBCL/ibrutinib was detected via immunohistochemistry (IHC), and the expression of PDGFD in DLBCL/ibrutinib-resistant strains and their parental counterparts were examined via reverse transcription-quantitative PCR and western blot analyses. Subsequently, a drug-resistant cell model of DLBCL/ibrutinib in which PDGFD was silenced was constructed. The apoptosis of the DLBCL/ibrutinib-resistant strains was examined using MTT and flow cytometry assays. EGFR gene expression was then assessed. At the same time, a PDGFD-interfering plasmid and an EGFR overexpression plasmid were transfected into the DLBCL drug-resistant cells (TMD8-ibrutinib, HBL1-ibrutinib) separately or together. MTT was used to measure cell proliferation and changes in the IC50 of ibrutinib. A total of 86 DEGs that increased in the CR, PR and SD tissues were screened, and then evaluated with GO and KEGG. The interaction network diagram showed that there was a regulatory relationship between PDGFD and disease-related genes, and that PDGFD could indirectly target the ibrutinib target gene EGFR, indicating that PDGFD could regulate DLBCL via EGFR. IHC results showed high expression of PDGFD in diffuse large B-cell lymphoma tissues with ibrutinib tolerance. PDGFD expression in ibrutinib-resistant DLBCL cells was higher compared with in parental cells. Following interference with PDGFD expression in ibrutinib-resistant DLBCL cells, the IC50 value of ibrutinib decreased, the rate of apoptosis increased and EGFR expression decreased. In brief, EGFR overexpression can reverse the resistance of DLBCL to ibrutinib via PDGFD interference, and PDGFD induces the resistance of DLBCL to ibrutinib via EGFR.

This study sought to assess whether the objective response (OR, including complete response and partial response) of first-line chemotherapy can predict overall survival (OS) for patients with metastatic triple-negative breast cancer (mTNBC) in both clinical trial and a real-world setting. The survival predictable parameters were assessed in two independent cohorts, the training cohort of 236 patients as part of a phase 3 trial (CBCSG006, Trial registration number NCT0128762) and the validation cohort of 360 patients from the real-world setting. Univariable and multivariable Cox proportional hazard models were applied to explore associations with progression-free survival and OS in the training cohort and then in the validation cohort. OR (OR vs non-OR, HR, 0.438, p

Background CBCSG006 trial reported the superior efficacy of cisplatin plus gemcitabine (GP) regimen than paclitaxel plus gemcitabine (GT) regimen as first-line treatment of metastatic triple-negative breast cancer (mTNBC). This study focused on the updated survival data and the explorations of potential biomarkers for efficacy. Patients and methods Germ-line mutations of homologous recombination (HR) panel, BRCA1/2 included, were evaluated in 55.9% (132/236) patients. PD-L1 expression was evaluated in 48.3% (114/236) patients. A nonparametric sliding-window subpopulation treatment effect pattern plot (STEPP) methodology was used to analyze the absolute survival benefits. All statistical tests were two-sided. Results Median progression-free survival (PFS) was 7.73 [95% confidence interval (CI) 6.46–9.00] months for GP arm and 6.07 (95% CI 5.32–6.83) months for GT arm (P = 0.005). No significant difference in overall survival (OS) was observed. There was significant interaction between HR status and treatment for PFS and status of HR deficient significantly correlated with higher objective response rate (ORR) and longer PFS in GP arm than in GT arm (71.9% versus 38.7%, P = 0.008; 10.37 versus 4.30 months, P = 0.011). There was no significant interaction between germ-line BRCA1/2 (gBRCA1/2) status and treatment for PFS. Patients with gBRCA1/2 mutation had numerically higher ORR and prolonged PFS in GP arm than in GT arm (83.3% versus 37.5%, P = 0.086; 8.90 versus 3.20 months, P = 0.459). There was no significant interaction between PD-L1 status and treatment for PFS, and no significant differences in ORR, PFS or OS between two arms regardless of PD-L1 status. In STEPP analysis, patients with lower composite risks had more absolute benefits in PFS than those with higher composite risks. Conclusions GP regimen has superior efficacy than GT regimen as first-line chemotherapy for mTNBC patients. Germ-line mutations of BRCA1/2 and HR panel are possible biomarkers for better performance of cisplatin-based regimens. A composite risk model was developed to guide patient selection for GP treatment in TNBC patients. Trial registration ClinicalTrials.gov, NCT01287624.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with limited treatment options and poor prognosis. Gemcitabine plus carboplatin (GC) is one of the preferred chemotherapeutic regimens for patients with metastatic triple-negative breast cancer(mTNBC), with a median progression-free survival(PFS) of 4.6 months in the first-line patients (O'Shaughnessy J, et al. J Clin Oncol 2014). Gemcitabine plus cisplatin (GP) has also demonstrated promising efficacy and safety in the first-line phase III trial of mTNBC, with a median PFS of 7.7 months (Hu XC, Lancet Oncol 2015). A recent analysis, based on data derived from a cohort of 379 mTNBC patients, indicated that patients receiving cisplatin-based regimen as the first-line chemotherapy showed better PFS compared with other platinum agents (8.0 vs 4.3 months, P = 0.03) (Zhang J, et al. Oncotarget 2015). To further investigate the superiority between carboplatin and cisplatin when combined with gemcitabine, this phase II study was conducted to directly compare the efficacy and safety of GP with GC in the first-line treatment for patients with metastatic triple negative breast cancer. Trial Design: This prospective phase II, single center, open-label, randomized study has been designed to compare the efficacy and safety of GP with GC as the first-line treatment for mTNBC. Patients are randomized 1:1 to receive gemcitabine (1250 mg/m2, D1,8) plus cisplatin (75 mg/m2, D1) or gemcitabine (1000mg/m2, D1,8) plus carboplatin (area under the curve 2 mg × min/mL, D1,8) every 21 days until disease progression or intolerable toxicity. Eligibility Criteria: Patients with histologically confirmed triple negative metastatic breast cancer, with no prior chemotherapy in metastatic setting will be included in this trial. Eligible patients must be between 18 and 70 years of age with a performance status of 0–1, adequate organ function and at least one RECIST 1.1-measurable lesion. Specific Aims: The primary endpoint is PFS. Secondary endpoints include objective response rate, safety and overall survival. Statistical Methods: The sample size of the present study was determined based on the results of two phase III clinical trials: the median PFS for patients receiving GC and GP as the first-line treatment for mTNBC was 4.6 and 7.7 months, respectively. This design was hypothesized that GP would be superior to GC in terms of efficacy. Thus, in order to detect an improvement of median PFS from 4.6 months to 7.7 months, with 80% power and a 1-sided type I error of 0.05, 136 patients would be required. Considering a drop-out rate of 10%, a total of 150 patients planned to be enrolled. Present Accrual and Target Accrual: Target accrual: 150. Present accrual (2017/5/24): 83 Contact information: Contact: Xichun Hu, MD, PHD xchu2009@hotmail.com Biyun Wang, MD pro_wangbiyun@163.com ClinicalTrials.gov Identifier: NCT02341911. Citation Format: Gong C, Cao E, Wang Z, Zhang J, Wang L, Cao J, Zhang S, Tao Z, Li T, Zhao Y, Li Y, Wang B, Hu X. A phase II randomized trial of gemcitabine plus cisplatin(GP) vs. gemcitabine plus carboplatin (GC) as the first-line treatment for patients with metastatic 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 OT2-05-01.