TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Low level of ARID1A contributes to adaptive immune resistance and sensitizes triple-negative breast cancer to immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) shed new light on triple-negative breast cancer (TNBC), but only a minority of patients demonstrate response. Therefore, adaptive immune resistance (AIR) needs to be further defined to guide the development of ICI regimens. METHODS: Databases, including The Cancer Genome Atlas, Gene Ontology Resource, University of California Santa Cruz Genome Browser, and Pubmed, were used to screen epigenetic modulators, regulators for CD8+ T cells, and transcriptional regulators of programmed cell death-ligand 1 (PD-L1). Human peripheral blood mononuclear cell (Hu-PBMC) reconstruction mice were adopted for xenograft transplantation. Tumor specimens from a TNBC cohort and the clinical trial CTR20191353 were retrospectively analyzed. RNA-sequencing, Western blotting, qPCR and immunohistochemistry were used to assess gene expression. Coculture assays were performed to evaluate the regulation of TNBC cells on T cells. Chromatin immunoprecipitation and transposase-accessible chromatin sequencing were used to determine chromatin-binding and accessibility. RESULTS: The epigenetic modulator AT-rich interaction domain 1A (ARID1A) gene demonstrated the highest expression association with AIR relative to other epigenetic modulators in TNBC patients. Low ARID1A expression in TNBC, causing an immunosuppressive microenvironment, promoted AIR and inhibited CD8+ T cell infiltration and activity through upregulating PD-L1. However, ARID1A did not directly regulate PD-L1 expression. We found that ARID1A directly bound the promoter of nucleophosmin 1 (NPM1) and that low ARID1A expression increased NPM1 chromatin accessibility as well as gene expression, further activating PD-L1 transcription. In Hu-PBMC mice, atezolizumab demonstrated the potential to reverse ARID1A deficiency-induced AIR in TNBC by reducing tumor malignancy and activating anti-tumor immunity. In CTR20191353, ARID1A-low patients derived more benefit from pucotenlimab compared to ARID1A-high patients. CONCLUSIONS: In AIR epigenetics, low ARID1A expression in TNBC contributed to AIR via the ARID1A/NPM1/PD-L1 axis, leading to poor outcome but sensitivity to ICI treatment.

Cuproptosis-related risk score predicts prognosis and characterizes the tumor microenvironment in colon adenocarcinoma.

Introduction: Cuproptosis is a novel copper-dependent regulatory cell death (RCD), which is closely related to the occurrence and development of multiple cancers. However, the potential role of cuproptosis-related genes (CRGs) in the tumor microenvironment (TME) of colon adenocarcinoma (COAD) remains unclear. Methods: Transcriptome, somatic mutation, somatic copy number alteration and the corresponding clinicopathological data of COAD were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO). Difference, survival and correlation analyses were conducted to evaluate the characteristics of CRGs in COAD patients. Consensus unsupervised clustering analysis of CRGs expression profile was used to classify patients into different cuproptosis molecular and gene subtypes. TME characteristics of different molecular subtypes were investigated by using Gene set variation analysis (GSVA) and single sample gene set enrichment analysis (ssGSEA). Next, CRG Risk scoring system was constructed by applying logistic least absolute shrinkage and selection operator (LASSO) cox regression analysis and multivariate cox analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) were used to exam the expression of key Risk scoring genes. Results: Our study indicated that CRGs had relatively common genetic and transcriptional variations in COAD tissues. We identified three cuproptosis molecular subtypes and three gene subtypes based on CRGs expression profile and prognostic differentially expressed genes (DEGs) expression profile, and found that changes in multilayer CRGs were closely related to the clinical characteristics, overall survival (OS), different signaling pathways, and immune cell infiltration of TME. CRG Risk scoring system was constructed according to the expression of 7 key cuproptosis-related risk genes (GLS, NOX1, HOXC6, TNNT1, GLS, HOXC6 and PLA2G12B). RT-qPCR and IHC indicated that the expression of GLS, NOX1, HOXC6, TNNT1 and PLA2G12B were up-regulated in tumor tissues, compared with those in normal tissues, and all of GLS, HOXC6, NOX1 and PLA2G12B were closely related with patient survival. In addition, high CRG risk scores were significantly associated with high microsatellite instability (MSI-H), tumor mutation burden (TMB), cancer stem cell (CSC) indices, stromal and immune scores in TME, drug susceptibility, as well as patient survival. Finally, a highly accurate nomogram was constructed to promote the clinical application of the CRG Risk scoring system. Discussion: Our comprehensive analysis showed that CRGs were greatly associated with TME, clinicopathological characteristics, and prognosis of patient with COAD. These findings may promote our understanding of CRGs in COAD, providing new insights for physicians to predict prognosis and develop more precise and individualized therapy strategies.

Immune Checkpoint Inhibitors Combined with Targeted Therapy: The Recent Advances and Future Potentials.

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.

Analysis of clinical features, genomic landscapes and survival outcomes in HER2-low breast cancer.

BACKGROUND: Novel human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugates prompt the identification of the HER2-low subtype. However, the biological significance of HER2-low expression in breast cancer is unclear. METHODS: Clinical and genomic data of 579 metastatic breast cancer patients were reviewed from our next-generation sequencing (NGS) database and genomic analysis of early breast cancer patients from TCGA was also analyzed. FINDINGS: First, the clinicopathological characteristics of HER2-low patients were profoundly influenced by HR status and no difference of prognosis was observed between HER2-low and HER2-zero patients when paired by HR status, but notably HER2-low patients showed similar metastatic patterns to HER2-positive patients in the HR-positive (HR+ ) subgroup, with more brain and initial lung metastases and more cases of de novo stage IV breast cancer than HER2-zero patients. Second, among patients with primary HER2-low or HER2-zero tumors, the discordance of HER2 status between primary and metastatic tumors was significant, with 48.4% of patients with HER2-zero primary tumors exhibiting HER2-low phenotype in metastatic tumors in the HR+ subgroup. Third, within HR+ and HR-negative subtypes, HER2-low and HER2-zero tumors showed no substantial differences in mutation alterations and copy number variations. Forth, germline BRCA2 mutations were observed only in HER2-low patients in our NGS database, especially in HR+ HER2-low tumors. Finally, three molecular subtypes based on genomic alterations in HER2-low breast cancer were identified, which provided novel insights into heterogeneity in HER2-low breast cancer. CONCLUSIONS: After correcting for HR expression, only marginal differences in clinical and molecular phenotypes were determined between HER2-low and HER2-zero breast cancer. Therefore, HER2-low breast cancer is insufficient to be defined as a distinct molecular entity, but rather a heterogenous disease.

A study of clinical and molecular characteristics in bilateral primary breast cancer.

BACKGROUND: Bilateral primary breast cancer (BPBC) is a rare type of breast cancer. Studies on the clinicopathologic and molecular characteristics of BPBC in a metastatic context are very limited. METHODS: A total of 574 unselected metastatic breast cancer patients with clinical information were enrolled in our next-generation sequencing (NGS) database. Patients with BPBC from our NGS database were regarded as the study cohort. In addition, 1467 patients with BPBC and 2874 patients with unilateral breast cancer (UBC) from the Surveillance, Epidemiology, and End Results (SEER) public database were also analyzed to determine the characteristics of BPBC. RESULTS: Among the 574 patients enrolled in our NGS database, 20 (3.5%) patients had bilateral disease, comprising 15 (75%) patients with synchronous bilateral disease and 5 (25%) patients with metachronous bilateral disease. Eight patients had bilateral hormone receptor-positive (HR+)/human epidermal growth factor receptor-negative (HER2-) tumors, and three had unilateral HR+/HER2- tumors. More HR+/HER2- tumors and lobular components were found in BPBC patients than in UBC patients. The molecular subtype of the metastatic lesions in three patients was inconsistent with either side of the primary lesions, which suggested the importance of rebiopsy. Strong correlations in clinicopathologic features between the left and right tumors in BPBC were exhibited in the SEER database. In our NGS database, only one BPBC patient was found with a pathogenic germline mutation in BRCA2. The top mutated somatic genes in BPBC patients were similar to those in UBC patients, including TP53 (58.8% in BPBC and 60.6% in UBC) and PI3KCA (47.1% in BPBC and 35.9% in UBC). CONCLUSIONS: Our study suggested that BPBC may tend to be lobular carcinoma and have the HR+/HER2- subtype. Although our study did not find specific germline and somatic mutations in BPBC, more research is needed for verification.

Chromatin Remodelling Molecule ARID1A Determines Metastatic Heterogeneity in Triple-Negative Breast Cancer by Competitively Binding to YAP.

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.

Landscape of DNA damage response gene alterations in breast cancer: A comprehensive investigation.

BACKGROUND: DNA damage response (DDR) gene alterations are prevalent in breast cancer (BC) and important for treatment decisions. Intensive studies on DDR alterations in BC are still needed. METHODS: The authors included 438 patients with metastatic breast cancer from their next-generation sequencing database and 1091 patients with early-stage breast cancer from The Cancer Genome Atlas (TCGA) database in the analysis to characterize molecular alterations in the DDR pathway. RESULTS: Germline DDR mutations were more prevalent in younger patients and those with HER2-negative cancers. Tumors with germline DDR mutations more commonly had somatic DDR mutations, especially those with germline Fanconi anemia (FA) pathway mutations. Notably, 66.67% (four of six) of patients with germline PALB2 mutations had tumors that harbored somatic PALB2 mutations. No differences in prognosis were observed in patients with germline or tumor somatic DDR mutations compared to patients and tumors that were wild-type. Compared to early BC, the frequency of somatic DDR mutations in metastatic cancers was significantly higher (24.89% vs. 16.02%, p < .001). Higher tumor mutation burdens were observed in cancers with somatic DDR mutations, but not in cancers with germline DDR mutations. Furthermore, tumors with somatic DDR mutations showed an abundance of anticancer immunological phenotypes. Somatic FA and mismatch repair pathway mutations were associated with increased expression of immune checkpoint molecules. Although most DDR genes were significantly positively associated with expression of proliferation-related genes, PARP3 expression was negatively correlated with MKI67 expression. Lower PARP3 expression was associated with a worse prognosis in TCGA database by multivariate Cox analysis. CONCLUSIONS: Patients with germline FA mutations more frequently have tumors with somatic DDR mutations. Somatic DDR mutations lead to anticancer immunological phenotypes in BC. No differences in prognosis according to germline or somatic DDR mutations were found.

High-purity isolation platelets by gradient centrifugation plus filtration.

INTRODUCTION: Platelets can be used as a liquid biopsy source to provide rapid, up-to-date, and relevant information on tumor pathology and treatment response. However, there is still a lack of high efficiency methods for platelet isolation with high purity. METHODS: Three platelet isolation methods were evaluated by platelet recovery and purity. The platelet inhibition cocktail (PIC) was added into peripheral blood, or was not allowed to access the effect of the platelet activation. The CD61, CD45, and CD62P labelled platelets, leukocytes and activated platelets were detected by flow cytometry. Quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS) were employed to determine the gene expression levels. A time-dependent experiment combined with qPCR was used to determine the time limit for platelet isolation at room temperature. RESULTS: Compared to the gradient centrifugation alone, and gradient centrifugation plus filtration and magnetic beads separation, gradient centrifugation plus filtration was the preferred method for more efficient and high-purity platelet isolation, with a recovery rate of 9.1% and a purity of 99.98%. Furthermore, there was no difference in platelet activation level, regardless of whether PIC was used. Moreover, the rate of platelet RNA degradation did not differ when platelets were isolated within 48 h of blood collection. CONCLUSION: Gradient centrifugation plus filtration at room temperature within 48 h of blood collection, without PIC, is a novel protocol with high recovery and purity rate to isolate platelets.

Identification of cuproptosis-related subtypes, construction of a prognosis model, and tumor microenvironment landscape in gastric cancer.

Introduction: Cuproptosis is a novel identified regulated cell death (RCD), which is correlated with the development, treatment response and prognosis of cancer. However, the potential role of cuproptosis-related genes (CRGs) in the tumor microenvironment (TME) of gastric cancer (GC) remains unknown. Methods: Transcriptome profiling, somatic mutation, somatic copy number alteration and clinical data of GC samples were downloaded from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database to describe the alterations of CRGs from genetic and transcriptional fields. Differential, survival and univariate cox regression analyses of CRGs were carried out to investigate the role of CRGs in GC. Cuproptosis molecular subtypes were identified by using consensus unsupervised clustering analysis based on the expression profiles of CRGs, and further analyzed by GO and KEGG gene set variation analyses (GSVA). Genes in distinct molecular subtypes were also analyzed by GO and KEGG gene enrichment analyses (GSEA). Differentially expressed genes (DEGs) were screened out from distinct molecular subtypes and further analyzed by GO enrichment analysis and univariate cox regression analysis. Consensus clustering analysis of prognostic DEGs was performed to identify genomic subtypes. Next, patients were randomly categorized into the training and testing group at a ratio of 1:1. CRG Risk scoring system was constructed through logistic least absolute shrinkage and selection operator (LASSO) cox regression analysis, univariate and multivariate cox analyses in the training group and validated in the testing and combined groups. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression of key Risk scoring genes. Sensitivity and specificity of Risk scoring system were examined by using receiver operating characteristic (ROC) curves. pRRophetic package in R was used to investigate the therapeutic effects of drugs in high- and low- risk score group. Finally, the nomogram scoring system was developed to predict patients' survival through incorporating the clinicopathological features and CRG Risk score. Results: Most CRGs were up-regulated in tumor tissues and showed a relatively high mutation frequency. Survival and univariate cox regression analysis revealed that LIAS and FDX1 were significantly associated with GC patients' survival. After consensus unsupervised clustering analysis, GC patients were classified into two cuproptosis molecular subtypes, which were significantly associated with clinical features (gender, age, grade and TNM stage), prognosis, metabolic related pathways and immune cell infiltration in TME of GC. GO enrichment analyses of 84 DEGs, obtained from distinct molecular subtypes, revealed that DEGs primarily enriched in the regulation of metabolism and intracellular/extracellular structure in GC. Univariate cox regression analysis of 84 DEGs further screened out 32 prognostic DEGs. According to the expression profiles of 32 prognostic DEGs, patients were re-classified into two gene subtypes, which were significantly associated with patients' age, grade, T and N stage, and survival of patients. Nest, the Risk score system was constructed with moderate sensitivity and specificity. A high CRG Risk score, characterized by decreased microsatellite instability-high (MSI-H), tumor mutation burden (TMB) and cancer stem cell (CSC) index, and high stromal and immune score in TME, indicated poor survival. Four of five key Risk scoring genes expression were dysregulated in tumor compared with normal samples. Moreover, CRG Risk score was greatly related with sensitivity of multiple drugs. Finally, we established a highly accurate nomogram for promoting the clinical applicability of the CRG Risk scoring system. Discussion: Our comprehensive analysis of CRGs in GC demonstrated their potential roles in TME, clinicopathological features, and prognosis. These findings may improve our understanding of CRGs in GC and provide new perceptions for doctors to predict prognosis and develop more effective and personalized therapy strategies.

Comparison of two regimens of weekly paclitaxel plus gemcitabine in patients with metastatic breast cancer: propensity score-matched analysis of real-world data.

Purpose: Weekly gemcitabine + paclitaxel (wGT) administration is widely applied in real-world clinical practice. The 28-day and 21-day regimens of wGT are the most widely accepted regimens. We evaluated the efficacy and safety of wGT administration in patients with metastatic breast cancer (MBC) and compared the two regimens. Methods: Patients with human epidermal growth factor receptor 2 (HER-2)-negative MBC who received wGT between October 2013 and October 2016 were identified using an electronic database. The outcome variables included progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and safety profile. Propensity score matching was performed to minimize potential confounders. Results: A total of 140 patients were included. The median PFS and OS was 7.8 [95% confidence interval (CI) = 7.0-8.7] months and 22.5 (95% CI = 18.8-26.1) months, respectively. The toxicity of wGT was manageable. Among the patients, 90 (64.3%) received the 21-day regimen and 50 (35.7%) received the 28-day regimen. A higher number of younger patients and patients receiving later-line therapy received the 28-day regimen. There was no significant difference between the two groups in PFS after propensity score matching, though subgroup analysis showed that patients with early relapse benefited more from the 28-day regimen. The ORR was numerically higher in 28-day regimen (37.8% versus 28.0%, p = 0.310). However, the 21-day regimen was better tolerated than the 28-day regimen. Conclusion: wGT administration showed efficacy and safety in patients with MBC. The efficacy was comparable between the two regimens after adjustment for confounding factors while the 21-day regimen was better tolerated. Plain Language Summary: 21-day regimen of wGT was well tolerated in patients with metastatic breast cancer Weekly gemcitabine + paclitaxel (wGT) administration is widely applied in real-world clinical practice. The 28-day and 21-day regimens of wGT are the most widely accepted regimens. We evaluated the efficacy and safety of wGT administration in patients with metastatic breast cancer (MBC) and compared the two regimens. Patients with human epidermal growth factor receptor 2 (HER-2)-negative MBC who received wGT between October 2013 and October 2016 were identified using an electronic database. The outcome variables included progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and safety profile. Propensity score matching was performed to minimize potential confounders. We found that the efficacy was comparable between the two regimens after adjustment for confounding factors while the 21-day regimen was better tolerated.

Apatinib plus vinorelbine versus vinorelbine for metastatic triple-negative breast cancer who failed first/second-line treatment: the NAN trial.

While therapies such as chemotherapy combined with immunotherapy, sacituzumab govitecan, and PARP inhibitors are available for metastatic TNBC, on disease progression after these therapies, the mainstay of therapy is chemotherapy. Apatinib is a small-molecule tyrosine kinase inhibitor that has promising anti-angiogenesis and antitumor activity for TNBC. We aimed to evaluate the safety and efficacy of adding apatinib to chemotherapy in patients with advanced TNBC with failed first/second-line treatment. A total of 66 patients were randomly assigned, in a 1:1 ratio, to receive vinorelbine or vinorelbine with apatinib in 28-day cycles. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), overall response rate (ORR) and safety. 33 received apatinib plus vinorelbine and 32 received vinorelbine (1 was withdrawal). Median PFS was significantly longer in the apatinib plus vinorelbine group than in the vinorelbine group (3.9 months vs. 2.0 months; hazard ratio, 1.82; 95% confidence interval [CI], 1.06 to 3.11; P = 0.026). Median OS was 11.5 months with apatinib plus vinorelbine and 9.9 months with vinorelbine (HR,1.01; 95% CI, 0.51 to 1.97; P = 0.985). The ORR was 9.1% in the apatinib plus vinorelbine group and 6.3% in the vinorelbine group (P = 0.667). The most common treatment-related hematologic grade 3-4 adverse events in apatinib plus vinorelbine group, were leukopenia, granulocytopenia, anemia, and thrombocytopenia. no treatment-related nonhematologic grade 4 adverse events or treatment-related deaths were observed. Collectively, adding apatinib to vinorelbine shows a promising benefit in PFS compared to vinorelbine monotherapy, with an excellent toxicity profile, warranting further exploration.

Phase 1b clinical trial of pucotenlimab (HX008), a novel anti-PD-1 monoclonal antibody, combined with gemcitabine and cisplatin in the first-line treatment of metastatic triple-negative breast cancer.

Background: Pucotenlimab, also called HX008, is a humanized anti-PD-1 antagonist IgG4 mAb. It blocks programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1), and programmed death ligand-2 (PD-L2). In the CBCSG 006 trial, gemcitabine plus cisplatin (GP) has shown impressive antitumor activity as first-line therapy for metastatic triple-negative breast cancer (mTNBC). The phase 1b study was conducted to assess the safety and preliminary antitumor activity of pucotenlimab when combined with GP in patients with mTNBC in the first-line setting. Methods: Eligible patients with mTNBC with ≥6 months of DFI (disease-free interval) who have never received antitumor therapy for metastatic disease were screened. Participants received pucotenlimab at 3 mg/kg (d1, q3w) plus gemcitabine at 1,250 mg/m2 (d1, 8, q3w) and cisplatin at 75 mg/m2 (d1, q3w). Eligible patients received up to six cycles of pucotenlimab along with GP chemotherapy, while pucotenlimab could be maintained until disease progression or unacceptable toxicity occurred or withdrawal of informed consent. This study was registered in China under registration number CTR20191353. Results: Between July 2019 and March 2020, 31 patients were enrolled in this study. The median age was 50 (range 28-68) years. Among 31 patients who were evaluated, 25 (80.6%) experienced objective response and the other six (19.4%) experienced stable disease (SD). As of 4 August, the median progression-free survival (PFS) was 9.0 months (95% CI, 6.2-9.2). The most common grade 3 or 4 treatment-related adverse events included neutropenia (74.1%), anemia (35.5%), thrombocytopenia (32.3%), hypocalcemia (9.7%), hypokalemia (9.7%), and alanine aminotransferase increased (6.5%). There were no treatment-related deaths. Conclusion: Pucotenlimab plus GP demonstrated promising activity and a manageable safety profile in patients with mTNBC in the first-line setting.

Temporal Heterogeneity of HER2 Expression and Spatial Heterogeneity of 18F-FDG Uptake Predicts Treatment Outcome of Pyrotinib in Patients with HER2-Positive Metastatic Breast Cancer.

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.

Mammary adipocytes protect triple-negative breast cancer cells from ferroptosis.

Ferroptosis, a novel non-apoptotic form of cell death, can induce tumor cell death and treatment resistance. Lipid metabolism is closely related to ferroptosis; however, the effect of mammary adipocytes on breast cancer ferroptosis remains to be elucidated. Here, we established the co-culture system of adipocyte-breast cancer cells and revealed the protection of triple-negative breast cancer from ferroptosis by adipocytes. Then, we performed the lipidomics analysis comparing lipid metabolites of co-cultured and normal-cultured cells. Mechanistically, oleic acid secreted from adipocytes inhibited lipid peroxidation and ferroptosis of triple-negative breast cancer cells in the presence of ACSL3. Taken together, mammary adipocytes can protect breast cancer cells from ferroptosis through oleic acid in the presence of ACSL3. These findings could provide new ideas and targets for tumor treatment.

Exosomal MMP-1 transfers metastasis potential in triple-negative breast cancer through PAR1-mediated EMT.

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. 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. 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, 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. 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.

Dalpiciclib Combined With Pyrotinib and Letrozole in Women With HER2-Positive, Hormone Receptor-Positive Metastatic Breast Cancer (LORDSHIPS): A Phase Ib Study.

Purpose: The LORDSHIPS study aimed to explore the safety and efficacy of a novel fully oral triplet combination of dalpiciclib (a potent cyclin-dependent kinase 4/6 inhibitor), pyrotinib (a HER2 tyrosine kinase inhibitor) and endocrine therapy letrozole in patients with HER2-positive, hormone receptor (HR)-positive metastatic breast cancer (MBC) in the front-line setting. Patients and Methods: Postmenopausal women with HER2-positive, HR-positive MBC were recruited in the dose-finding phase Ib trial. A standard 3 + 3 design was used to determine safety, tolerability, and recommended phase II dose (RP2D) for the combination. Results: A total of 15 patients were enrolled to three dose combination cohorts (letrozole/pyrotinib/dalpiciclib, level/I: 2.5/400/125 mg, n=5; level/L1: 2.5/400/100 mg, n=6; level/L2: 2.5/320/125 mg, n=4). Three patients experienced dose-limiting toxicities (level/I, n=2; level/L1, n=1) and level/L2 was identified as RP2D. The most frequent grade 3-4 adverse events were neutropenia (46.7%), leukopenia (40.0%), oral mucositis (26.7%) and diarrhea (20.0%). The confirmed objective response rate (ORR) was 66.7% (95% CI: 38.4% to 88.2%). The confirmed ORR of study treatment as first line (1L) and second line (2L) HER2-targeted therapy was 85.7% (6/7) and 50.0% (4/8), respectively. Median progression-free survival (PFS) was 11.3 months (95% CI: 5.3 months to not reached). PFS in 1L setting was not reached yet, while PFS in 2L setting was 10.9 months (95% CI: 1.8 to 13.7 months). Conclusions: The fully oral combination of dalpiciclib, pyrotinib and letrozole is a promising chemotherapy-sparing treatment option for HER2-positive, HR-positive MBC patients. The planned dose-expansion phase II study is ongoing. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03772353.

Efficacy and safety of mitoxantrone hydrochloride liposome injection in Chinese patients with advanced breast cancer: a randomized, open-label, active-controlled, single-center, phase II clinical trial.

PURPOSE: 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.

Phase II trail of nab-paclitaxel in metastatic breast cancer patients with visceral metastases.

BACKGROUND: Visceral metastases account for 48-67% of metastatic breast cancer (MBC) patients and presage a worse overall survival. Previous study suggested potential effect of nab-paclitaxel on patients with visceral metastases subgroups. This phase II trial was conducted to explore the efficacy and safety of nab-paclitaxel in such a high-risk group of patients. METHODS: In this prospective, single-center, open-label, phase II study, MBC patients with visceral metastases (N = 80) received nab-paclitaxel (Abraxane, 125 mg/m2, D1, D8, D15 every 28 days). RESULTS: The median PFS was 5.1 months (95% CI: 4.2-6.0 months), with an ORR of 33.8% (95% CI 21.3-43.8%) and CBR of 66.2% (95% CI 56.3-75.0%). In univariate analysis, patients with premenopausal status had a trend of better treatment outcome. Multivariate analysis demonstrated non brain metastasis (adjusted HR 0.31, 95% CI 0.12-0.83, P = 0.019) and first line treatment (adjusted HR 0.37, 95% CI 0.17-0.81, P = 0.013) as independent predictors of longer PFS. The overall safety was acceptable with most common treatment-related, grade ≥ 3 toxicities of neutropenia (16.3%) and sensory neuropathy (3.7%). CONCLUSIONS: This phase II trial documented satisfactory efficacy and safety of nab-paclitaxel in MBC patients with visceral metastases, providing evidence for relative clinical practice. Patients in first line therapy had better treatment outcome. For patients with premenopausal status or brain metastasis, further alternatives (for example, combined chemotherapy or targeting therapy) might be required. This study also demonstrated the efficacy and safety of 125 mg/m2 nab-paclitaxel among Asian patients. TRIAL REGISTRATION: This research is registered under clinicaltrials.gov (NCT02687490, February 22, 2016).

Profiling Receptor Tyrosine Kinase Fusions in Chinese Breast Cancers.

BACKGROUND: Receptor tyrosine kinases (RTKs) are a class of tyrosine kinases that regulate cell-to-cell communication and control a variety of complex biological functions. Dysregulation of RTK signaling partly due to chromosomal rearrangements leads to novel tyrosine kinase fusion oncoproteins that are possibly driver alterations to cancers. Targeting some RTK fusions with specific tyrosine kinases inhibitors (TKIs) is an effective therapeutic strategy across a spectrum of RTK fusion-related cancers. However, there is still a paucity of extensive RTK fusion investigations in breast cancer. This study aims to characterize RTK fusions in Chinese breast cancer patients. METHODS: An in-house DNA sequencing database of 1440 Chinese breast cancer patients with a capture-based panel (520 gene or 108 gene-panel) was thoroughly reviewed. A total of 2,229 samples including 1,045 tissues and 1,184 plasmas were analyzed. RTK fusion was defined as an in-frame fusion with the tyrosine kinase domain of the RTK completely retained. Concomitant mutations were also analyzed and tumor mutational burden (TMB) was calculated. Patients' clinical characteristics were retrieved from case records. RESULTS: A total of 30 RTK fusion events were identified from 27 breast cancer patients with a prevalence of 1.875%%. FGFR2 fusions were seen the most commonly (n=7), followed by RET (n=5), ROS1 (n=3), NTRK3 (n=3), BRAF (n=2), and NTRK1 (n=2). Other RTK fusions including ALK, EGFR, FGFR1, FGFR3, MET, and NTRK2 were identified in one patient each. A total of 27 unique resultant fusion proteins (22 with a novel partner) were discovered including 19 intrachromosomal rearrangements and 8 interchromosomal ones. Twenty-one fusions had the tyrosine kinase domain in-frame fused with a partner gene and six were juxtaposed with an intergenic space. Among the 27 fusions, FGFR2-WDR11 (E17: intergenic) (n=3) and ETV6-NTRK3 (E5:E15) (n=2) occurred recurrently. Of note, the normalized abundance of RTK fusion (fusion AF/max AF) correlated negatively with TMB (r=-0.48, P=0.017). Patients with TMB < 8 (Mutations/Mb) displayed a higher fusion abundance than those with TMB ≥ 8 (Mutations/Mb) (P=0.025). Moreover, CREBBP mutation only co-occurred with FGFR2 fusion (P=0.012), while NTRK3 fusion and TP53 mutation were mutually exclusive (P=0.019). CONCLUSION: This is the first study comprehensively delineating the prevalence and spectrum of RTK fusions in Chinese breast cancers. Further study is ongoing to identify the enriched subpopulation who may benefit from RTK fusion inhibitors.