NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis.

Breast cancer (BC) poses a great threat to women's health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

Assessment of the impact of residual tumors at different sites post-neoadjuvant chemotherapy on prognosis in breast cancer patients and development of a disease-free survival prediction model.

Background: Residual disease after neoadjuvant chemotherapy (NAC) in breast cancer patients predicts worse outcomes than pathological complete response. Differing prognostic impacts based on the anatomical site of residual tumors are not well studied. Objectives: The study aims to assess disease-free survival (DFS) in breast cancer patients with different residual tumor sites following NAC and to develop a nomogram for predicting 1- to 3-year DFS in these patients. Design: A retrospective cohort study. Methods: Retrospective analysis of 953 lymph node-positive breast cancer patients with residual disease post-NAC. Patients were categorized into three groups: residual disease in breast (RDB), residual disease in lymph nodes (RDN), and residual disease in both (RDBN). DFS compared among groups. Patients were divided into a training set and a validation set in a 7:3 ratio. Prognostic factors for DFS were analyzed to develop a nomogram prediction model. Results: RDB patients had superior 3-year DFS of 94.6% versus 85.2% for RDN and 81.8% for RDBN (p < 0.0001). Clinical T stage, N stage, molecular subtype, and postoperative pN stage were independently associated with DFS on both univariate and multivariate analyses. Nomogram integrating clinical tumor-node-metastasis (TNM) stage, molecular subtype, pathological response demonstrated good discrimination (C-index 0.748 training, 0.796 validation cohort), and calibration. Conclusion: The location of residual disease has prognostic implications, with nodal residuals predicting poorer DFS. The validated nomogram enables personalized DFS prediction to guide treatment decisions.

Understanding the impact of residual tumor location on prognosis after breast cancer treatment After receiving neoadjuvant chemotherapy, a treatment to shrink tumors before surgery, some breast cancer patients may still have residual tumor cells. Our study focuses on how the location of these remaining tumors ­ whether in the breast, lymph nodes, or both ­ affects the likelihood of the cancer not returning within the next 1 to 3 years. This likelihood is known as 'disease-free survival' (DFS). We analyzed data from 953 breast cancer patients who underwent neoadjuvant chemotherapy and still had residual tumors. By comparing DFS among patients with tumors remaining in different locations, we discovered that the specific location of the residual tumor significantly impacts the patient's long-term health and recovery. Additionally, we developed a predictive tool called a 'nomogram' to help doctors and patients assess the risk of cancer recurrence in the next 1 to 3 years. This tool considers various factors such as the size and type of the tumor, as well as the location and extent of the residual tumor after chemotherapy. Our research offers new insights into understanding the risk of recurrence after breast cancer treatment. This work not only enhances our comprehension of breast cancer management but also aids in devising more personalized and effective treatment strategies for patients in the future.

Targeted axillary dissection after neoadjuvant chemotherapy for highly selective patients with initial cN1 breast cancer: A single-center prospective trial.

BACKGROUND: Sentinel lymph node (SLN) biopsy is gradually accepted as the standard of care in breast cancer patients with down-staged axillary disease after neoadjuvant chemotherapy (NAC). However, it is still difficult to precisely define pre-NAC clinical node-positive (cN1) and post-NAC clinical node-negative (ycN0). This prospective single-center trial was designed to evaluate the feasibility and accuracy of standard targeted axillary dissection (TAD) after NAC in highly selective pre-NAC cN1 patients (not considering ultrasound-based axillary ycN staging). METHODS: This prospective trial included patients with initial pre-NAC cT1-3N1M0 invasive breast cancer but with a rigorous definition of cN1 from the Affiliated Cancer Hospital of Zhengzhou University. When NAC was effective (including complete and partial responses) and preoperative axillary palpation was negative, preoperative ultrasound-based axillary staging was not considered, and all patients underwent TAD followed by axillary lymph node (LN) dissection. The detection rate (DR) and false-negative rate (FNR) of TAD were calculated. RESULTS: A total of 82 patients were included, and 77 of them were eligible for data analysis. The DR for TAD was 94.8% (73/77). There were 26 patients with one abnormal LN at the time of diagnosis based on ultrasound, 45 patients with two, and 2 patients with three. One patient had one TAD LN, four patients had two TAD LNs, and 68 patients had three or more TAD LNs. Preoperative axillary palpation yielded negative results for all 73 patients who successfully underwent TAD. Preoperative ultrasound-based ycN0 and ycN+ conditions were detected for 52 and 21 cases, respectively. The FNR was 7.4% (2/27) for standard TAD (≥3 SLNs), which was lower than that of all successful TAD (≥1 SLN; 10.0%, 3/30). CONCLUSIONS: In rigorously defined pre-NAC cN1 breast cancer patients, standard TAD is feasible for those with negative axillary palpation after NAC, and FNR is also less than 10%. REGISTRATION: chictr.org.cn , ChiCTR2100049093.

The Impact of Sentinel Lymph Node Biopsy on Female Patients With T3-4c Breast Cancer and 1-2 Positive Lymph Nodes: A Population-Based Cohort Study.

PURPOSE: This study aimed to evaluate the efficacy of sentinel lymph node biopsy (SLNB) in patients diagnosed with cT3-4c breast cancer with no more than 2 positive sentinel lymph nodes. METHODS: Using data from the Surveillance, Epidemiology, and End Results (SEER) database, this retrospective study identified patients diagnosed with T3-4c breast cancer between 2010 and 2015. These patients were then categorized into 2 groups: the SLNB group, which underwent examination of 1-5 regional lymph nodes and the axillary lymph node dissection (ALND) group, which underwent examination of ≥10 regional lymph nodes. Propensity score matching analysis was used to assess the efficacy of SLNB in cT3-4c patients. RESULTS: A total of 1139 patients were included in the analysis, with 423 and 716 patients in the SLNB and ALND groups, respectively. The 10-year overall survival (OS) and breast cancer-specific survival (BCSS) rates in the SLNB group were 66.1% and 76.3%, respectively, compared with 66.0% and 73.8%, respectively. Statistical analysis revealed no significant differences between the 2 groups in terms of OS (HR = 1.00, 95% CI = 0.80-1.25, P = .997) and BCSS (HR = 1.08, 95% CI = 0.83-1.41, P = .551). Even after 1:1 propensity score matching, there were no significant differences in OS (HR = 0.87, 95% CI = 0.65-1.16, P = .341) and BCSS (HR = 0.82, 95% CI = 0.59-1.16, P = .266) between the 2 groups. CONCLUSION: This study demonstrates that SLNB does not adversely affect the survival of cT3-4c breast cancer patients with 1-2 sentinel lymph node metastases.

Neoadjuvant camrelizumab plus nab-paclitaxel and epirubicin in early triple-negative breast cancer: a single-arm phase II trial.

Immunotherapy combined with chemotherapy has been demonstrated to be effective in early triple-negative breast cancer (TNBC). In this single-arm, phase II study with Simon's two-stage design, we investigated the efficacy and safety of neoadjuvant camrelizumab plus chemotherapy in patients with early TNBC (NCT04213898). Eligible female patients aged 18 years or older with histologically confirmed treatment-naïve early TNBC were treated with camrelizumab (200 mg, on day 1), nab-paclitaxel (125 mg/m2, on days 1, 8, and 15), and epirubicin (75 mg/m2, on day 1) every three weeks for six cycles. The primary end point was the pathological complete response; secondary endpoints included safety, objective response rate, and long-term survival outcomes of event-free survival, disease-free survival, and distant disease-free survival. A total of 39 patients were enrolled between January 2020 and October 2021. Twenty-five patients achieved a pathological complete response (64.1%, 95%CI: 47.2, 78.8). The objective response rate was 89.7% (95%CI: 74.8, 96.7), including 35 patients with partial responses. Treatment-related adverse events of grade 3 or 4 occurred in 30 (76.9%) patients. In conclusion, the trial meets the prespecified endpoints showing promising efficacy and manageable safety of neoadjuvant camrelizumab plus nab-paclitaxel and epirubicin chemotherapy in female patients with early TNBC. Long-term survival outcomes are still pending.

A Nomogram for Identifying HR+/Her2- Breast Cancer Patients with Positive Sentinel Lymph Nodes and Omitted Axillary Lymph Node Dissection Who Need Abemaciclib Therapy.

BACKGROUND The efficacy of abemaciclib in high-risk patients with early-stage HR+/Her2- breast cancer has been verified by MonarchE. However, accurately determining the number of axillary lymph node (ALN) metastases remains challenging. The Z0011 trial changed the axillary management strategy, eliminating the need for axillary lymph node dissection (ALND) in patients with 1-2 sentinel lymph node (SLN) metastases. Therefore, further exploration is needed to identify patients who could benefit from abemaciclib therapy. MATERIAL AND METHODS This retrospective study included cT1-2N0M0 HR+/Her2- patients with 1-2 positive SLNs who underwent ALND. Clinicopathological data were collected, and logistic regression analyses identified independent predictors for ≥4 positive ALNs. A predictive nomogram was developed, and discrimination and calibration were evaluated using the C-index and calibration curve. Clinical efficacy was assessed using decision curve analysis (DCA). RESULTS We enrolled 444 patients, with 77 (17.3%) having ≥4 positive ALNs. Independent predictors for ≥4 positive ALNs included abnormal ALN on ultrasound, mammographic calcifications, T stage, and the number of positive SLNs. The nomogram demonstrated an AUC of 0.777 (95% CI: 0.735-0.815, P<0.001), and internal validation showed good calibration and discrimination (C-index, 0.802; 95% CI: 0.779-0.824). DCA revealed a positive net benefit for risk levels ranging from 5% to 54%. CONCLUSIONS This nomogram is a convenient and reliable tool to predict the risk of ≥4 positive ALNs in HR+/Her2- patients. It aids in protocol selection by identifying SLN-positive patients who may benefit from abemaciclib therapy without ALND.

Identification of a novel glycolysis-related prognosis risk signature in triple-negative breast cancer.

Introduction: Triple-negative breast cancer (TNBC) is a particularly aggressive cluster of breast cancer characterized by significant molecular heterogeneity. Glycolysis is a metabolic pathway that is significantly associated with cancer progression, metastasis, recurrence and chemoresistance. However, the potential roles of glycolysis-related genes in TNBC remain unclear. Methods: In the present study, we identified 108 glycolysis-related differentially expressed genes (DEGs) between breast cancer (BRCA) tumor tissues and normal tissues, and we divided patients into two different clusters with significantly distinct molecular characteristics, clinicopathological features, prognosis, immune cell infiltration and mutation burden. We then constructed a 10-gene signature that classified all TNBCs into low- and high-risk groups. Results: The high-risk group had significantly lower survival than the low-risk group, which implied that the risk score was an independent prognostic indicator for TNBC patients. Consequently, we constructed and validated a prognostic nomogram, which accurately predicted individual overall survival (OS) of TNBC. Moreover, the risk score predicted the drug sensitivity of chemotherapeutic agents and immunotherapy for TNBC patients. Discussion: The present comprehensive analysis of glycolysis-related DEGs in TNBC provides new methods for prognosis prediction and more effective treatment strategies.

Neoadjuvant Efficacy of Three Targeted Therapy Strategies for HER2-Positive Breast Cancer Based on the Same Chemotherapy Regimen.

(1) Background: The objective of our study was to provide evidence for choosing the optimal neoadjuvant therapy strategies for patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. Three neoadjuvant targeted therapy strategies (H + Py, trastuzumab plus pyrotinib; H, trastuzumab; HP, trastuzumab plus pertuzumab) based on the same chemotherapy regimen (TC, docetaxel and carboplatin) were included in the present study; (2) Methods: We retrospectively analyzed patients with HER2-positive breast cancer who were treated with neoadjuvant TCH + Py, TCH or TCHP, followed by surgery. The outcome was the pathological complete response (pCR) rate; (3) Results: In total, 545 patients were enrolled. The pCR rate was 55.6% (35/63) in the TCH + Py cohort, 32.7% (93/284) in the TCH cohort, and 56.6% (112/198) in the TCHP cohort. The multivariate analysis showed that patients who received TCH had less possibility to achieve pCR than those who received TCH + Py (odds ratio (OR) = 0.334, 95% confidence interval (CI): 0.181−0.619, p < 0.001), while patients who received TCHP had comparable possibility to those who received TCH + Py (OR = 1.043, 95%CI: 0.554−1.964, p = 0.896); (4) Conclusions: TCH + Py provides a better pCR rate compared with TCH, and a comparable pCR rate with TCHP among patients with HER2-positive breast cancer in the neoadjuvant setting. The present study supports a novel potential treatment option for these patients. Further studies need to be explored in the future.

Pathological response and predictive role of tumour-infiltrating lymphocytes in HER2-positive early breast cancer treated with neoadjuvant pyrotinib plus trastuzumab and chemotherapy (Panphila): a multicentre phase 2 trial.

PURPOSE: Panphila evaluated pyrotinib plus trastuzumab, docetaxel and carboplatin as neoadjuvant therapy for early breast cancer (BC), and investigated the predictive role of immune cell subpopulations. PATIENTS AND METHODS: In this multicentre phase 2 study, patients with human epidermal growth factor receptor 2-positive, stage T2-3N0-3M0 BC received pyrotinib 400 mg once daily plus docetaxel (75 mg/m2, day 1), carboplatin (6 mg/mL/min, day 1) and trastuzumab (8 mg/kg loading dose and 6 mg/kg maintenance dose, day 1) for 6 cycles of 21 days each. Simon's 2-stage design was adopted. The primary end-point was pathological complete response (pCR, ypT0/is ypN0) rate. Tumour-infiltrating lymphocytes (TILs) were assessed by haematoxylin and eosin staining and multiplex immunohistochemistry. RESULTS: In the modified intention-to-treat population (n = 69), 38 patients (55.1%) achieved pCR. In the safety population (n = 74), the most common grade ≥3 adverse events were diarrhoea (43.2%), anaemia (37.8%), vomiting (16.2%) and platelet count decrease (10.8%). No treatment-related deaths occurred. Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by stromal (s)-CD20+, s-CD8+ and s-CD4+ TILs. Unsupervised hierarchical clustering of stromal immune markers identified a group of patients characterised by high s-CD20+, s-CD8+, s-CD4+ and s-FOXP3+ immune cells infiltration, which was independently associated with pCR. CONCLUSION: Neoadjuvant pyrotinib plus trastuzumab-based chemotherapy exhibits promising efficacy and manageable toxicity in patients with human epidermal growth factor receptor 2-positive early BC, and thus phase 3 trials are warranted. Our findings also contribute to understanding the potential role of the immune microenvironment in response to neoadjuvant pyrotinib-based therapy.

A novel 12-gene prognostic signature in breast cancer based on the tumor microenvironment.

Background: The progression of breast cancer (BC) is highly dependent on the tumor microenvironment. Inflammation, stromal cells, and the immune landscape have been identified as significant drivers of BC in multiple preclinical studies. Therefore, this study aimed to clarify the predictive relevance of stromal and immune cell-associated genes in patients suffering from BC. Methods: We employed the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) algorithm to calculate the stromal and immunological scores, which were then used to evaluate differentially expressed genes (DEGs) in BC samples using The Cancer Genome Atlas (TCGA) database. Univariate analyses were conducted to identify the DEGs linked to survival in BC patients. Next, the prognostic DEGs (with a log-rank P<0.05) were used to create a risk signature, and the least absolute shrinkage and selection operator (LASSO) regression method was used to analyze and optimize the risk signature. The following formula was used to compute the prognostic risk score values: Risk score = Gene 1 * ß1 + Gene 2 * ß2 +… Gene n * ßn. The median prognostic risk score values were used to divide BC patients into the low-risk (LR) and high-risk (HR) groups. The patient samples of the validation cohort were then assessed using this formula. We used principal component analysis (PCA) to determine the expression patterns of the different patient groups. Gene Set Enrichment Analysis (GSEA) was used to determine whether there were significant variations between the groups in the evaluated gene sets. Results: The present study revealed that DEGs linked with survival were closely associated with immunological responses. A prognostic signature was constructed that consisted of 12 genes (ASCL1, BHLHE22, C1S, CLEC9A, CST7, EEF1A2, FOLR2, KLRB1, MEOX1, PEX5L, PLA2G2D, and PPP1R16B). According to their survival, BC patients were separated into LR and HR groups using the identified 12-gene signature. The immunological status and immune cell infiltration were observed differently in the LR and HR groups. Conclusions: Our results provide novel insights into several microenvironment-linked genes that influence survival outcomes in patients with BC, which suggests that these genes could be candidate therapeutic targets.

Pyrotinib plus capecitabine for patients with human epidermal growth factor receptor 2-positive breast cancer and brain metastases (PERMEATE): a multicentre, single-arm, two-cohort, phase 2 trial.

BACKGROUND: Patients with HER2-positive metastatic breast cancer have a high risk of developing brain metastases. Efficacious treatment options are scarce. We investigated the activity and safety of pyrotinib plus capecitabine in patients with HER2-positive metastatic breast cancer and brain metastases. METHODS: We did a multicentre, single-arm, two-cohort, phase 2 trial in eight tertiary hospitals in China. Patients aged 18 years or older who had radiotherapy-naive HER2-positive brain metastases (cohort A) or progressive disease after radiotherapy (cohort B), with an Eastern Cooperative Oncology Group performance status of 0-2, received pyrotinib 400 mg orally once daily, and capecitabine 1000 mg/m2 orally twice daily for 14 days, followed by 7 days off every 3 weeks until disease progression or unacceptable toxicity. The primary endpoint was confirmed intracranial objective response rate by investigator assessment according to the Response Evaluation Criteria In Solid Tumours (version 1.1). Activity and safety were analysed in patients with at least one dose of study drug. The study is ongoing, but recruitment is complete. The study is registered with ClinicalTrials.gov, NCT03691051. FINDINGS: Between Jan 29, 2019, and July 10, 2020, we enrolled 78 women: 51 (86%) of 59 patients in cohort A and 18 (95%) of 19 patients in cohort B had previous exposure to trastuzumab. Median follow-up duration was 15·7 months (IQR 9·7-19·0). The intracranial objective response rate was 74·6% (95% CI 61·6-85·0; 44 of 59 patients) in cohort A and 42·1% (20·3-66·5; eight of 19 patients) in cohort B. The most common grade 3 or worse treatment-emergent adverse event was diarrhoea (14 [24%] in cohort A and four [21%] in cohort B). Two (3%) patients in cohort A and three (16%) in cohort B had treatment-related serious adverse events. No treatment-related deaths occurred. INTERPRETATION: To our knowledge, this is the first prospective study showing the activity and safety of pyrotinib plus capecitabine in patients with HER2-positive breast cancer and brain metastases, especially in radiotherapy-naive population. This combination deserves further validation in a randomised, controlled trial. FUNDING: National Cancer Centre Climbing Foundation Key Project of China, Jiangsu Hengrui Pharmaceuticals. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Effect of the HER-2/CEP17 ratio in IHC 2+/FISH-amplified breast cancer on pathological complete response to neoadjuvant pertuzumab and trastuzumab treatment-a retrospective cohort study.

Background: Human epidermal growth factor receptor-2 (HER-2) protein expression level could serve as a predictor of pathological complete response (pCR) to neoadjuvant trastuzumab-containing regimens. The aim of the present study was to evaluate whether pCR to neoadjuvant trastuzumab and pertuzumab treatment is dependent on the level of the HER-2/CEP17 (chromosome enumeration probe 17) ratio in immunohistochemistry (IHC) 2+/fluorescence in situ hybridization (FISH)-amplified breast cancer. Methods: Patients with primary IHC 2+/FISH-amplified breast cancer who underwent neoadjuvant anti-HER-2 dual-targeted therapies were retrospectively included between January 1, 2020 and May 30, 2021. The primary predictive variable was HER-2/CEP17 ratio, and the primary outcome variable was pCR. Other variables consisted of age, menopausal status, tumor-node-metastasis (TNM) stage, estrogen receptor (ER), progesterone receptor (PgR), and Ki-67. Association between clinicopathologic variables and pCR was evaluated using the chi-square test and logistic regression analysis. Results: The median age of the patients was 51.78 years (25-67 years), and 50.7% of the patients were in the premenopausal stage. The clinical stage at diagnosis was Stage III in 38 patients (55.1%). Of all patients, 40.6% patients were estrogen receptor positive, and 75.4% patients had a Ki-67 index of ≥30%. The overall pCR (ypN0/isypN0) rate was 31.9%. Patients with HER-2/CEP17 ratio ≥6.0 had a pCR rate of 55.0%, it was statistically higher than 22.4% in patients with HER-2/CEP17 ratio <6.0. Logistic regression analysis confirmed the independent association between HER-2/CEP17 ratio and pCR (P=0.020, OR: 5.203, 95% CI: 1.302-20.783). Conclusions: A HER-2/CEP17 ratio ≥6.0 might be related to more achievement of pCR in the neoadjuvant anti-HER-2 dual-targeted therapies. Further studies are needed to validate the finding.

Establishment of a model for predicting sentinel lymph node metastasis in early breast cancer based on contrast-enhanced ultrasound and clinicopathological features.

BACKGROUND: Sentinel lymph node (SLN) biopsy (SLNB) is the standard procedure for axillary staging in clinically node-negative (cN0) breast cancer patients. However, the positive rate of SLNs among cN0 stage patients is 26-35%. The identification of appropriate candidates for SLNB is quite challenging. This study aimed to establish and verify a predictive model of SLN metastasis using contrast-enhanced ultrasound (CEUS) and other clinicopathological indicators. METHODS: The clinicopathological data of 224 patients who had undergone SLNB at the Affiliated Cancer Hospital of Zhengzhou University from June 2018 to July 2019 were analyzed retrospectively. The risk prediction model of SLN metastasis was established by logistic regression analysis. According to the ß value of each variable in the model, a risk score system of SLN metastasis was established and verified using the internal population. The predictive model was prospectively applied to 73 patients from July 2019 to September 2019 to evaluate the clinical value of the model in patients with early breast cancer. RESULTS: Multivariate analysis confirmed that body mass index (BMI), SLN aspect ratio of CEUS mode, SLN aspect ratio of mammography, lympho-vascular invasion, and cytokeratin (CK)5/6 were independent risk factors for SLN metastasis. A scoring system was established according to the above risk factors, and a receiver operating characteristic (ROC) curve was drawn. After internal- and external verification, a corrected ROC curve was drawn, respectively. The ROC curve of the modeling group, internal verification group, and external verification group was 0.9075 (95% CI: 0.8616-0.9534), 0.8766 (95% CI: 0.8192-0.9341), and 0.8505 (95% CI: 0.7333-0.9676), respectively. CONCLUSIONS: We constructed and verified a prediction model of SLN metastasis in early breast cancer. The model has a specific predictive value for preoperative evaluation of SLN status.

Establishment and Verification of a Predictive Model for Node Pathological Complete Response After Neoadjuvant Chemotherapy for Initial Node Positive Early Breast Cancer.

OBJECTIVE: Axillary node status after neoadjuvant chemotherapy (NCT) in early breast cancer patients influences the axillary surgical staging procedure. This study was conducted for the identification of the likelihood of patients being node pathological complete response (pCR) post NCT. We aimed to recognize patients most likely to benefit from sentinel lymph node biopsy (SLNB) following NCT and to reduce the risk of missed detection of positive lymph nodes through the construction and validation of a clinical preoperative scoring prediction model. METHODS: The existing data (from March 2010 to December 2018) of the Chinese Society of Clinical Oncology Breast Cancer Database (CSCO-BC) was used to evaluate the independent related factors of node pCR after NCT by Binary Logistic Regression analysis. A predictive model was established according to the score of considerable factors to identify ypN0. Model performance was confirmed in a cohort of NCT patients treated between January 2019 and December 2019 in Henan Cancer Hospital, and model discrimination was evaluated via assessing the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Multivariate regression analysis showed that the node stage before chemotherapy, the expression level of Ki-67, biologic subtype, and breast pCR were all independent related factors of ypN0 after chemotherapy. According to the transformation and summation of odds ratio (OR) values of each variable, the scoring system model was constructed with a total score of 1-5. The AUC for the ROC curves was 0.715 and 0.770 for the training and the validation set accordingly. CONCLUSIONS: A model was established and verified for predicting ypN0 after chemotherapy in newly diagnosed cN+ patients and the model had good accuracy and efficacy. The underlined effective model can suggest axillary surgical planning, and reduce the risk of missing positive lymph nodes by SLNB after NCT. It has great value for identifying initial cN+ patients who are more appropriate for SLNB post-chemotherapy.

Predictive risk factors for sentinel lymph node metastasis using preoperative contrast-enhanced ultrasound in early-stage breast cancer patients.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is the standard procedure for axillary staging in clinically node-negative (cN0) breast cancer patients. The positive rate of SLNs in cN0 stage patients ranges from 20.5% to 25.5%, so identifying appropriate candidates for SLNB is quite challenging. The aims of this study were to assess whether contrast-enhanced ultrasound (CEUS) could be utilized to noninvasively predict SLN metastasis, and to explore the predictive value of the involved factors. METHODS: Between May 2016 and May 2018, 217 consenting breast cancer patients undergoing SLNB were enrolled. Before the surgery, CEUS was utilized to identify the SLNs, and predict whether metastasis had occurred according to their enhancement pattern. Blue dye was also used to identify the SLNs during SLNB. The rates of identification and accuracy of both methods were recorded. The predictive outcomes of SLNs identified by CEUS were recorded and compared with the pathological diagnosis. RESULTS: Of the 217 cases, SLNs in 212 cases were successfully identified, comprising 208 cases identified by CEUS and 206 cases by blue dye, with no significant difference between the two methods (P=0.6470). A total of 78 cases were predicted SLN-positive preoperatively by CEUS, comprising 61 cases of SLN metastasis confirmed by pathology and 17 cases of no SLN metastasis, and 130 cases were predicted SLN-negative by CEUS, comprising 6 cases of SLN metastasis and 124 cases of no SLN metastasis. The sensitivity of CEUS preoperative prediction was 91.0%, the specificity was 87.9%, the positive and negative predictive values were 78.2% and 95.4%, respectively, and the accuracy was 88.9%. The maximum diameter size of positive SLNs predicted by CEUS was greater than that of negative SLNs (mean value 1.67±0.06 vs. 1.40±0.05 cm, P=0.0007). Similarly, the primary tumor size predicted SLN-positive by CEUS was greater than that in patients with negative SLNs (mean value 2.64±0.12 vs. 1.79±0.09 cm, P<0.0001). CONCLUSIONS: CEUS accurately identified SLNs and can be used to noninvasively predict SLN metastasis in early-stage breast cancer patients. However, the primary tumor size and the SLN size should not be overlooked by clinicians when judging the status of SLNs. This novel method may be a recommended strategy for identifying appropriate SLNB candidates.

ATF2 inhibits ani-tumor effects of BET inhibitor in a negative feedback manner by attenuating ferroptosis.

BET inhibitor (BETi) has potential therapeutic effects on human cancer especially in breast cancer. However, the detailed mechanisms remain unclear. Herein, we found that BETi JQ1 and I-BET-151 (I-BET) activated ATF2 through JNK1/2 pathway in breast cancer cells MDA-MB-231 (MB-231). In addition, overexpression of ATF2 blocked the reduction of cell viability induced by JQ1 or I-BET in breast cancer MB-231 and BT-549 cells, cervical cancer HeLa cells and lung cancer A549 cells. The induction of cell death by BETi was also attenuated by ATF2 in MB-231 and BT-549 cells. By contrast, depletion of ATF2 increased cancer cell sensitivity to BETi. In MB-231 cells xenograft model, ATF2 significantly inhibited the anti-tumor effects of JQ1. By detection of the oxidized form gluthione, malondialdehyde and lipid ROS, we showed that overexpression of ATF2 inhibited ferroptosis induced by BETi, whereas depletion of ATF2 promoted ferroptosis by BETi. Furthermore, the underlying mechanisms of ATF2-reduced ferroptosis were investigated. Overexpressed and depleted ATF2 were found to significantly upregulate and downregulate NRF2 protein and mRNA expression, respectively. The significantly positive correlations between NRF2 and ATF2 gene expression were found in breast, lung and cervical cancer tissues from TCGA database. In NRF2-depleted MB-231 cells, ATF2 failed to attenuate JQ1-stimulated ferroptosis. All these results suggested that ATF2 inhibited BETi-induced ferroptosis by increasing NRF2 expression. Altogether, our findings illustrated ATF2 suppressed ani-tumor effects of BETi in a negative feedback manner by attenuating ferroptosis. BETi combined with ATF2 or NRF2 inhibitor might be a novel strategy for treatment of human cancer.

HN1L promotes migration and invasion of breast cancer by up-regulating the expression of HMGB1.

Recent reports showed that haematological and neurological expressed 1-like (HN1L) gene participated in tumorigenesis and tumour invasion. However, the expression and role of HN1L in breast cancer remain to be investigated. Here, bioinformatics, western blot and immunohistochemistry were used to detect the expression of HN1L in breast cancer. Wound healing, transwell assay, immunofluorescence assay and mass spectrum were used to explore the role and mechanism of HN1L on the migration and invasion of breast cancer, which was confirmed in vivo using a nude mice model. Results showed that HN1L was significantly over-expressed in breast cancer tissues, which was positively correlated with M metastasis of breast cancer patients. Silencing HN1L significantly inhibited the invasion and metastasis of breast cancer cells in vitro and lung metastasis in nude mice metastasis model of breast cancer. Mechanistically, HN1L interacted with HSPA9 and affected the expression of HMGB1, playing a key role in promoting the invasion and metastasis of breast cancer cell. These results suggested that HN1L was an appealing drug target for breast cancer.

Knockdown of LINC00665 inhibits proliferation and invasion of breast cancer via competitive binding of miR-3619-5p and inhibition of catenin beta 1.

BACKGROUND: Long intergenic non-protein coding RNA00665 (LINC00665) plays a crucial tumorigenic role in many cancers, such as gastric cancer and lung adenocarcinoma. However, its role and mechanism of action in the progression of breast cancer (BC) are unknown. METHODS: LINC00665 expression levels were determined using quantitative polymerase chain reaction analysis with BC tissues and cell lines. BC cell proliferation was tested by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, whereas BC cell migration and invasion capabilities were analyzed by performing transwell migration assays. Percentages of apoptotic cells were measured by flow cytometry. Interactions between LINC00665 and miR-3169-5p were examined by performing luciferase reporter assays, and the expression levels of proteins, such as ß-catenin, were examined by western blot analysis. RESULTS: LINC00665 was expressed at high levels in BC tissues and cells. Upregulated LINC00665 expression correlated with tumor size and tumor, node, and metastasis stages, but not with the age of patients. LINC00665 knockdown inhibited BC cell proliferation, migration, and invasion, whereas it promoted apoptosis. Moreover, bioinformatics analysis and the luciferase reporter assay revealed that LINC00665 bound the microRNA (miR) miR-3619-5p. miR-3619-5p expression correlated negatively with LINC00665 expression in BC tissues. miR-3619-5p overexpression inhibited BC cell proliferation, migration, and invasion, but promoted apoptosis. Simultaneous knockdown of LINC00665 and miR-3619-5p led to increased cell proliferation, migration, and invasion, and inhibited apoptosis. Additionally, catenin beta 1, which encodes the ß-catenin protein, was the target gene of miR-3619-5p. ß-catenin expression clearly decreased after LINC00665 knockdown and miR-3619-5p overexpression, but increased after simultaneous knockdown of LINC00665 and miR-3619-5p. CONCLUSION: LINC00665 knockdown inhibited BC cell proliferation and invasion by binding miR-3619-5p and inhibiting ß-catenin expression.

Macrophages confer resistance to BET inhibition in triple-negative breast cancer by upregulating IKBKE.

BET inhibitors (BETi) exhibit a strong anti-tumor activity in triple-negative breast cancer (TNBC). However, BETi resistance has been reported in TNBC. The mechanisms of resistance have not been demonstrated. Tumor-associated macrophages (TAMs) are frequently involved in cancer cells resistance to chemotherapy, also associated with poor prognosis in TNBC. However, the role of TAMs in BETi resistance remains unknown. Here, we found that BETi JQ1 and I-BET151 exerted anti-tumor effects in TNBC by decreasing IKBKE expression to attenuate NF-κB signaling. TAMs have been reported to associate with chemoresistance in breast cancer. Here, we firstly found that TNBC-stimulated TAMs activated NF-κB signaling by upregulating IKBKE expression to enhance breast cancer cells resistance to BETi. The IKBKE levels were also proved to be higher in clinical TNBC tissues than Non-TNBC tissues, suggesting feedback induction of IKBKE expression by TNBC-stimulated TAMs in TNBC. Moreover, the induction of IKBKE by TAMs in TNBC cells was identified to be associated with STAT3 signaling, which was activated by TAM-secreted IL-6 and IL-10. Lastly, the combination of inhibitors of BET and STAT3 exerted a synergistic inhibition effects in TAM-cocultured or TAM CM-treated TNBC cells in vitro and in vivo. Altogether, our findings illustrated TNBC-activated macrophages conferred TNBC cells resistance to BETi via IL-6 or IL-10/STAT3/IKBKE/NF-κB axis. Blockade of IKBKE or double inhibition of BET and STAT3 might be a novel strategy for treatment of TNBC.

NR5A2 synergizes with NCOA3 to induce breast cancer resistance to BET inhibitor by upregulating NRF2 to attenuate ferroptosis.

BET inhibitors (BETi) exert an excellent anti-cancer activity in breast cancer. However, the identification of new potential targets to enhance breast cancer sensitivity to BETi is still an enormous challenge. Both NR5A2 and NCOA3 are frequently involved in cancer cells resistance to chemotherapy, also associated with poor prognosis in breast cancer. However, the functions of NR5A2 and NCOA3 in BETi resistance remains unknown. In this study, we found that BETi JQ1 and I-BET151 exhibited anti-cancer effects in breast cancer by inducing ferroptosis. NCOA3 as a coactivator synergized with NR5A2 to prevent BETi-induced ferroptosis. Mechanistically, we identified NR5A2 synergized with NCOA3 to increase expression of NRF2, a transcription factor that controls the expression of many antioxidant genes. Moreover, inhibition of NR5A2 or NCOA3 using small molecule inhibitors enhanced anti-cancer effects of BETi against breast cancer in vivo and in vitro. Altogether, our findings illustrated NR5A2 synergized with NCOA3 to confer breast cancer cells resistance to BETi by induction of NRF2. Inhibition of NR5A2/NCOA3 combined with BETi might be a novel strategy for treatment of breast cancer.