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Triple-negative breast cancer (TNBC) is a heterogeneous disease and lacks effective treatment. Our previous study classified TNBCs into four subtypes with putative therapeutic targets. Here, we report the final results of FUTURE, a phase II umbrella trial designed to explore whether the subtyping-based strategy may improve the outcomes in metastatic TNBC patients. A total of 141 patients with a median of three previous lines of therapies in the metastatic setting were enrolled in seven parallel arms. Confirmed objective responses were achieved in 42 patients (29.8%; 95% confidence interval [CI], 22.4–38.1). The median values of progression-free survival and overall survival were 3.4 (95% CI: 2.7–4.2) and 10.7 (95% CI: 9.1–12.3) months, respectively. Given Bayesian predictive probability, efficacy boundaries were achieved in four arms. Furthermore, integrated genomic and clinicopathological profiling illustrated associations of clinical and genomic parameters with treatment efficacy, and the efficacy of novel antibody–drug conjugates was explored in preclinical TNBC models of subtypes for which treatment was futile. In general, the FUTURE strategy recruits patients efficiently and provides promising efficacy with manageable toxicities, outlining a direction for further clinical exploration.

Background: The effect of neoadjuvant therapy on tumor downstaging and breast-conserving during surgery is well documented. Pathologic complete response (pCR) after neoadjuvant treatment was associated with long-term survival. HLX02 (Zercepac®), a biosimilar of trastuzumab, showed the same efficacy, safety, and immunogenicity as the reference drug in human epidermal growth factor receptor-2 (HER2)-positive patients. Despite this, real-world evidence of its effectiveness when combined with pertuzumab in neoadjuvant treatment of HER-2 positive breast cancer still lacks. Methods: In this retrospective real-world study, women with confirmed invasive HER2-positive breast cancer who have received chemotherapy plus HLX02 and pertuzumab (Perjeta®) as neoadjuvant therapy were enrolled. Patients must be over 18 years old, have an Eastern Cooperative Oncology Group performance status score of 0 or 1, and have a baseline left ventricular ejection fraction of ≥ 55%. Patients without pathological assessment after neoadjuvant therapy were excluded. Pathologic complete response (pCR) was defined as no residual invasive tumors in mammary glands and axillary lymph nodes. Clinical response was assessed using RECIST1.1. To investigate the factors associated with pCR, univariate and multivariate logistic regression (forward stepwise) analyses were conducted. Results: A total of 85 patients were enrolled in this study, and 55 patients (64.71%) achieved pCR after neoadjuvant therapy. There were 84 (98.82%) patients with partial response (PR), one (1.18%) patient with stable disease (SD). According to the univariable analysis, when compared to those with a tumor diameter ≤ 5 cm, patients with a tumor diameter > 5 cm at baseline showed a lower pCR rate (odds ratio [OR] = 0.286; 95% confidence interval [CI]: 0.108-0.758, P = 0.01). Patients with preoperative PR positivity > 10% showed lower pCR rate than those with preoperative PR positivity < 1% (OR = 0.115, 95% CI 0.036-0.372, P = 0.02). Besides, pCR was more common in patients with preoperative hormone receptor (HR)-negative than in those with preoperative HR-positive (ER or PR positivity > 10%) (OR = 4.452, 95% CI 1.679-11.804, P < 0.01). Multivariable analyses showed that patients with tumor diameter > 5 cm had a lower pCR rate than those with tumor diameter ≤ 5cm (OR = 0.213; 95% CI 0.070-0.644, P = 0.01). Patients with preoperative HR-negative tumors were more likely to achieve pCR than those with preoperative HR-positive tumors (OR =5.649, 95% CI 1.927-16.556, P < 0.01) (Table 1). The treatment were well tolerated by patients, and no additional adverse events were reported. Conclusion: According to this real-world study, HLX02 in combination with pertuzumab as neoadjuvant therapy for HER2-positive breast cancer patients showed a similar pCR rate to that of dual-target neoadjuvant therapy reported in previous clinical trials. The treatment showed an encouraging effectiveness, and may become a novel neoadjuvant option for patients with HER2-positive breast cancer. The study was supported by the Natural Science Foundation of Shanghai (21ZR1414700). Table 1. Logistic regression for pCR. Citation Format: Yin Liu, Wen-Jia Zuo, Ruo-Xi Wang, Zhong-Hua Wang, Zhi-Ming Shao. Trastuzumab (HLX02) plus Pertuzumab as Dual-target Neoadjuvant Therapy for HER2-positive Breast Cancer: A Real-World Study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-20.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and lacks effective treatment. Our previous study classified TNBCs into four subtypes (luminal androgen receptor [LAR], immunomodulatory [IM], basal-like immune-suppressed [BLIS], mesenchymal-like [MES]) with distinct molecular features. We aimed to assess the efficacy and safety of molecular subtype-derived precision treatment in patients with heavily pretreated metastatic TNBC. Methods: This open-label, phase 2, umbrella trial included patients from four centers in China. Participants were women (aged ≥18 years) with histologically confirmed metastatic TNBC with disease progression after multiple lines of standard chemotherapy. Patients were enrolled into seven parallel arms according to their molecular subtypes: LAR with or without ERBB2 somatic mutation/amplification assigned to arm A (pyrotinib with capecitabine) and arm B (androgen inhibitor included therapy); IM assigned to arm C (anti-PD-1 antibody with nab-paclitaxel); BLIS with or without BRCA1/2 germline mutation assigned to arms D (PARP inhibitor included therapy) and E (anti-VEGFR included therapy); MES without or with PI3K-AKT mutation assigned to arms F (anti-VEGFR included therapy) and G (everolimus with nab-paclitaxel). Bayesian predictive probability was adopted to monitor each arm, which can be terminated independently according to a prespecified futility or efficacy boundary. This trial is registered with ClinicalTrials.gov, NCT03805399. Findings: Between October 18, 2018, and February 11, 2022, we enrolled 141 patients. All patients were heavily pretreated and resistant to six categories of the most common chemotherapeutic agents used in breast cancer treatment, with a median of 3 previous lines of therapies in the metastatic setting (Table 1 and 2). The median follow-up was 18.3 months (IQR 11.7-27.7). A confirmed objective response was achieved in 42 (29.8%, 95% CI 22.4-38.1) of the 141 patients. The median PFS was 3.4 months (95% CI 2.7-4.2), and the median OS was 10.7 months (95% CI 9.0-12.3) (Table 3). Arms A, C, E and G achieved efficacy boundaries, with 3 (75.0%) out of 4 patients in arm A, 20 (43.5%) out of 46 patients in arm C, 13 (28.3%) out of 46 patients in arm E, and 3 (33.3%) out of 9 patients in arm G achieving objective responses. Potential predictive biomarkers of efficacy in each arm were explored. Safety data were consistent with the known safety profiles of relevant drugs. Interpretation: We demonstrate the feasibility and clinical utility of a subtyping-based, genomic sequencing-guided strategy which allows the majority of heavily pretreated metastatic TNBCs to benefit from precision treatment. Most arms exhibit promising efficacy and manageable toxicities, providing subtyping schema to optimize personalized treatment. Table 1. The FUTURE trial schema. Patients are stratified into seven arms using the FUSCC 484-gene NGS panel testing and IHC subtyping. Abbreviations: mTNBC, metastatic triple-negative breast cancer; NGS, next-generation sequencing; IHC, immunohistochemistry; FUSCC, Fudan University Shanghai Cancer Center; LAR, luminal androgen receptor; IM, immunomodulatory; BLIS, basal-like immune-suppressed; MES, mesenchymal-like; n, number; AR, androgen receptor; PD-1, programmed cell death-1; PARPi, poly ADP-ribose polymerase inhibitor; VEGF, vascular endothelial growth factor; mTORi, mammalian target of rapamycin inhibitors. Table 2. Patient characteristics in the FUTURE trial. Table 3. Summary of treatment efficacy of TNBC in the FUTURE trial Citation Format: Zhi-Ming Shao, Zhong-Hua Wang, Yi-Zhou Jiang, Yin Liu, Xiu-Zhi Zhu, Yi Xiao, Song-Yang Wu, Wen-Jia Zuo, Qiang Yu, A-Yong Cao, Jun-Jie Li, Ke-Da Yu, Guang-Yu Liu, Jiong Wu, Tao Sun, Jiuwei Cui, Zheng Lv, Hui-Ping Li, Xiao-Yu Zhu. Subtyping-based platform guides precision medicine for heavily pretreated metastatic triple-negative breast cancer: a multicenter, phase 2, umbrella, FUTURE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-27-01.

Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor 2 (HER2) may be an alternative mechanism to HER2 activation and can affect the sensitivity toward HER2-targeted therapies. We aimed to investigate the prevalence, clinicopathologic characteristics, and functional relevance of novel HER2 mutations in breast cancer.Experimental Design: We performed Sanger sequencing of all exons of the HER2 gene in 1,248 primary tumors and 18 paired metastatic samples. Novel HER2 mutations were functionally characterized.Results: The total HER2 somatic mutation rate was 2.24% (28/1,248). Of the seven novel HER2 mutations, L768S and V773L were only detected in HER2-negative tumors, whereas K753E was found in HER2-positive disease. L768S and V773L mutations exhibited a significant increase in tyrosine kinase–specific activity and strongly increased the phosphorylation of signaling proteins in various cell lines. Xenograft experiments showed that NIH3T3 cells bearing the L768S and V773L mutations displayed more rapid growth. MCF10A, BT474, and MDA-MB-231 cells bearing the K753E mutation were resistant to lapatinib, but could be inhibited by neratinib. Finally, comparison of HER2 mutations in 18 pairs of primary and metastatic lesions revealed that the drug-resistant HER2 mutations (K753E and L755S) were enriched in metastatic lesions.Conclusions: HER2-negative breast cancer with activating mutations can benefit from HER2-targeted therapies. Meanwhile, mutations in the HER2 kinase domain might be a key mechanism of resistance to HER2-targeted therapy, and irreversible tyrosine kinase inhibitors such as neratinib may offer alternative treatment options. Clin Cancer Res; 22(19); 4859–69. ©2016 AACR.

Fig. S1. RNA-seq based RBP Library construction and CRISPR screen reveals RBP hits related to tumor growth and metastasis. Fig. S2. PHF5A is frequently up-regulated in breast cancer, associated with poor clinical outcome, and essential for cancer cell proliferation and migration. Fig. S3. PHF5A associates with the U2 snRNP complex and regulates complex stability. Fig. S4. PHF5A globally regulates splicing. Fig. S5. The PHD-like domain in PHF5A is required for its association with Histone H3. Fig. S6 PHF5A regulates FASTK splicing to antagonize apoptotic signaling in breast cancer cells. Table S1. Clinicopathologic Variables in 40 Cases of TNBC Patients. Table S2. Primers Used in RT-PCR and qPCR Analyses. Table S3. Gene List of a Census of 1,542 Manually Curated RBPs and 159 Library Contained RBPs. Table S4. sgRNA Sequence of Genes in RBP library. Table S5. RIGER Ranking of Screen Hits. Table S6. Correlation between Clinicopathologic Variables and Expression of PHF5A in 373 Cases of Breast Cancer. Table S7. Univariate and Multivariate Analysis of DFS. Table S8. Univariate and Multivariate Analysis of DFS in TNBC Subgroup.

Supplementary Figure S1. Flowchart of study design. Supplementary Figure S2. Protein structure visualizations of the HER2 somatic mutations. Supplementary Figure S3. HER2 mutations L768S and V773L induce HER2 internalization. Supplementary Figure S4. Kaplan-Meier estimates of relapse-free survival (RFS) according to HER2 mutation in different regions.

Supplementary Methods: Immunofluorescence

Supplementary Table S1. Sanger sequencing primers for all 27 exons of HER2 gene. Supplementary Table S2. Clinicopathologic characteristics of breast cancer patients enrolled in our study. Supplementary Table S3. Clinicopathologic characteristics of HER2-mutated breast cancer. Supplementary Table S4. Clinical information of patients with HER2-mutated breast cancer

Alternative splicing (AS) and its regulation play critical roles in cancer, yet the dysregulation of AS and its molecular bases in breast cancer development have not yet been elucidated. Using an in vivo CRISPR screen targeting RNA-binding proteins, we identified PHD finger protein 5A (PHF5A) as a key splicing factor involved in tumor progression. PHF5A expression was frequently upregulated in breast cancer and correlated with poor survival, and knockdown of PHF5A significantly suppressed cell proliferation, migration, and tumor formation. PHF5A was required for SF3b spliceosome stability and linked the complex to histones, and the PHF5A–SF3b complex modulated AS changes in apoptotic signaling. In addition, expression of a short truncated FAS-activated serine/threonine kinase (FASTK) protein was increased after PHF5A ablation and facilitated Fas-mediated apoptosis. This PHF5A-modulated FASTK–AS axis was widely present in breast cancer specimens, particularly those of the triple-negative subtype. Taken together, our findings reveal that PHF5A serves as an epigenetic suppressor of apoptosis and thus provides a mechanistic basis for breast cancer progression and may be a valuable therapeutic target.Significance: This study provides an epigenetic mechanistic basis for the aggressive biology of breast cancer and identifies a translatable therapeutic target. Cancer Res; 78(12); 3190–206. ©2018 AACR.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, and molecular subtyping may result in improved diagnostic precision and targeted therapies. Our previous study classified TNBCs into four subtypes with putative therapeutic targets. Here, we conducted the FUTURE trial (ClinicalTrials.gov identifier: NCT03805399), a phase Ib/II subtyping-based and genomic biomarker-guided umbrella trial, to evaluate the efficacy of these targets. Patients with refractory metastatic TNBC were enrolled and stratified by TNBC subtypes and genomic biomarkers, and assigned to one of these seven arms: (A) pyrotinib with capecitabine, (B) androgen receptor inhibitor with CDK4/6 inhibitor, (C) anti PD-1 with nab-paclitaxel, (D) PARP inhibitor included, (E) and (F) anti-VEGFR included, or (G) mTOR inhibitor with nab-paclitaxel. The primary end point was the objective response rate (ORR). We enrolled 69 refractory metastatic TNBC patients with a median of three previous lines of therapy (range, 1–8). Objective response was achieved in 20 (29.0%, 95% confidence interval (CI): 18.7%–41.2%) of the 69 intention-to-treat (ITT) patients. Our results showed that immunotherapy (arm C), in particular, achieved the highest ORR (52.6%, 95% CI: 28.9%–75.6%) in the ITT population. Arm E demonstrated favorable ORR (26.1%, 95% CI: 10.2%–48.4% in the ITT population) but with more high grade (≥ 3) adverse events. Somatic mutations of TOP2A and CD8 immunohistochemical score may have the potential to predict immunotherapy response in the immunomodulatory subtype of TNBC. In conclusion, the phase Ib/II FUTURE trial suggested a new concept for TNBC treatment, demonstrating the clinical benefit of subtyping-based targeted therapy for refractory metastatic TNBC.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by its highly aggressive behavior, early recurrence, and poor outcomes, when compared with other subtypes. Due to the absence of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, TNBC lacks meaningful biomarkers and an effective therapeutic strategy. Chemotherapy remains the main adjuvant treatment for patients with TNBC. Anthracycline/taxane-based regimens are the standard of care in adjuvant settings. The addition of capecitabine or platinum may offer extra benefits to patients with TNBC, but at the cost of increased toxicity or adverse events. Dose-dense chemotherapy may enhance treatment efficacy in patients who are able to tolerate the treatment regimen, especially in high-risk patients. As a heterogenous disease, TNBC can be classified into several molecular subtypes according to genomic or transcriptional features, which may indicate potential targets for more precise and individualized treatment strategies. With our increased understanding of signal pathways associated with TNBC, as well as the discovery of novel biomarkers indicative of TNBC prognosis, several new therapeutic options are under investigation, and some have already reported good results. In this review, we summarized the current conventional therapeutic strategies and emerging clinical trials regarding adjuvant treatment for TNBC. Furthermore, we evaluated the prognostic value of several potential targets and the progress of targeted therapy in TNBC, both in neoadjuvant and adjuvant settings.

BACKGROUND: Controversy remains regarding the predictive and prognostic value of serum human epidermal growth factor receptor 2 (HER2) in breast cancer. The purpose of this retrospective study was to determine the clinical utility and efficacy of serum HER2 (sHER2) in predicting treatment response and prognosis in patients with HER2-positive breast cancer undergoing neoadjuvant chemotherapy and trastuzumab treatment. METHODS: A total of 309 HER2-positive breast cancer patients diagnosed at Fudan University Shanghai Cancer Center from July 2015 to January 2019 were analyzed. Baseline sHER2 levels were obtained for all patients and sHER2 levels were collected after 2 cycles of treatment in 208 patients. A sHER2 level ≥15 ng/mL was regarded as “high expression” and sHER2

Background and PurposeAlthough tumor size and nodal status are the most important prognostic factors, it is believed that nodal status outperforms tumor size as a prognostic factor. In particular, when patients have a nodal stage greater than N2 (more than nine positive lymph nodes), it is well accepted that tumor size does not retain its prognostic value. Even in the newest American Joint Committee on Cancer (AJCC) prognostic staging system, which includes molecular subtype as an important prognostic factor, T1-3N2 patients are categorized as the same population. The same is true for T1-4N3 patients. Moreover, some physicians have speculated that for tumors staged N2 or greater, the smaller the tumor is, the more aggressive the tumor. Thus, this study aims to investigate the prognostic value of tumor stage (T stage) in patients with extensive nodal involvement and to compare the survival of T4N × M0 and T × N3M0.Patients and MethodsFemale breast cancer patients with nine or more positive lymph nodes or with T4 tumors were identified in the SEER registry between 2010 and 2015. The effect of T stage on breast cancer-specific survival (BCSS) was assessed using the Kaplan–Meier survival curve method and risk-adjusted Cox proportional hazard regression modeling. Survival comparison of T4NxM0 and TxN3M0 patients was also achieved using the Kaplan–Meier survival curve method and risk-adjusted Cox proportional hazard regression model.ResultsOverall, 21,696 women with N2-3 tumors were included from 284,073 patients.T stage, nodal stage (N stage), ER, PR, HER2 and grade were all independent prognostic factors (p ConclusionsIn patients with extensive nodal status, tumor stage remains a prognostic factor independent of other factors, such as ER, PR, HER2, and grade. In patients with T4Nx or TxN3 tumors, T4 tumors exhibit worse outcomes than N3 tumors independent of other prognostic factors. The AJCC staging system should be modified based on these findings.

Resistance to paclitaxel remains a major challenge in treating breast cancer. Our preclinical study suggested that TEKT4 germline variations in breast cancer are associated with paclitaxel resistance and increase vinorelbine sensitivity. This clinical trial compared the efficacy of paclitaxel and vinorelbine in breast cancer neoadjuvant chemotherapy. In this open-label, single-center, phase II trial, female patients with human epidermal growth factor receptor 2 (HER2)-negative, stage IIB–IIIC breast cancer harboring TEKT4 germline variations were randomly assigned to the paclitaxel plus epirubicin (PE) or vinorelbine plus epirubicin (NE). The primary endpoint was the pathologic complete response (pCR) rate, and the secondary endpoints were the objective response rate (ORR) and safety. Targeted sequencing of a panel comprising 484 breast-related genes was performed to identify pCR-associated somatic mutations in each group. 91 Patients were assigned to PE (46 patients) or NE (45 patients). NE numerically increased the pCR rate (22.2% versus 8.7%, P = 0.074). The ORRs for NE and PE were 82.2% and 76.1%, respectively. Interestingly, NE (15.4%) showed a significantly higher pCR rate than PE (0%) in the hormone receptor (HR)-positive subgroup (P = 0.044). Both regimens were well tolerated, with grade 3 and 4 toxicities reported at the expected levels. The biomarker analysis showed that UNC13D mutation predicted the pCR rate in NE (P = 0.011). Although the primary endpoint was not met, NE might bring clinical benefit to HR-positive patients or patients simultaneously carrying UNC13D mutations.

This study was designed to determine the safety and clinical efficacy of metronomic chemotherapy combined with aromatase inhibitors (AIs) for hormone receptor (HR)-positive advanced breast cancer (ABC) patients who cannot tolerate conventional-dose chemotherapy. Postmenopausal patients with HR-positive ABC, who exhibited disease progression after first-line AIs treatment and who could not tolerate or rejected conventional chemotherapy, were enrolled in this study. Patients received capecitabine 500 mg PO TID (could be reduced to 500 mg QD in case of adverse effects) and exemestane 25 mg QD (after PD with letrozole) or letrozole 2.5 mg QD (after PD with exemestane). The primary endpoints were safety and tolerance, the secondary endpoints were objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and time to treatment failure (TTF). In our analysis of 44 patients, the median age was 64 years (range 38–90) and 68.2% patients had at least two recurrences or metastatic lesions. Grade 3 toxicities (hand–foot syndrome) were observed only in 4 of the patients. Most patients exhibited no or mild toxicities. After a median follow-up of 14.8 months, ORR was 70.5%, CBR—77.3%, PFS—16.2 months, and TTF—14.4 months. Metronomic oral capecitabine combined with AIs showed good efficacy, minimal toxicities, and good tolerance in HR-positive patients with ABC. It is a potential treatment option especially for postmenopausal HR-positive ABC patients in poor general condition who cannot tolerate conventional chemotherapy. Trial registration: Clinicaltrials.gov NCT01924078.

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway contributes to the development and progression of tumors. Here, we determine that somatic mutations in PIK3CA (44%), PIK3R1 (17%), AKT3 (15%), and PTEN (12%) are prevalent and diverse in Chinese breast cancer patients, with 60 novel mutations identified. A high proportion of tumors harbors multiple mutations, especially PIK3CA plus PIK3R1 mutations (9.0%). Next, we develop a recombination-based mutation barcoding (ReMB) library for impactful mutations conferring clonal advantage in proliferation and drug responses. The highest-ranking PIK3CA and PIK3R1 mutations include previously reported deleterious mutations, as well as mutations with unknown significance. These PIK3CA and PIK3R1 impactful mutations exhibit a mutually exclusive pattern, leading to oncogenesis and hyperactivity of PI3K pathway. The PIK3CA impactful mutations are tightly associated with hormone receptor positivity. Collectively, these findings advance our understanding of PI3K impactful mutations in breast cancer and have important implications for PI3K-targeted therapy in precision oncology., The PI3K pathway is altered across various cancer types. Here the authors use amplicon exon sequencing to analyze the landscape of somatic mutations affecting the PI3K pathway specifically in breast cancer patients in China.

To investigate ovarian function and therapeutic efficacy among estrogen receptor (ER)-positive, premenopausal breast cancer patients treated with gonadotropin-releasing hormone agonist (GnRHa) and chemotherapy simultaneously or sequentially. This study was a phase 3, open-label, parallel, randomized controlled trial (NCT01712893). Two hundred sixteen premenopausal patients (under 45 years) diagnosed with invasive ER-positive breast cancer were enrolled from July 2009 to May 2013 and randomized at a 1:1 ratio to receive (neo)adjuvant chemotherapy combined with sequential or simultaneous GnRHa treatment. All patients were advised to receive GnRHa for at least 2 years. The primary outcome was the incidence of early menopause, defined as amenorrhea lasting longer than 12 months after the last chemotherapy or GnRHa dose, with postmenopausal or unknown follicle-stimulating hormone and estradiol levels. The menstrual resumption period and survivals were the secondary endpoints. The median follow-up time was 56.9 months (IQR 49.5–72.4 months). One hundred and eight patients were enrolled in each group. Among them, 92 and 78 patients had complete primary endpoint data in the sequential and simultaneous groups, respectively. The rates of early menopause were 22.8% (21/92) in the sequential group and 23.1% (18/78) in the simultaneous group [simultaneous vs. sequential: OR 1.01 (95% CI 0.50–2.08); p = 0.969; age-adjusted OR 1.13; (95% CI 0.54–2.37); p = 0.737]. The median menstruation resumption period was 12.0 (95% CI 9.3–14.7) months and 10.3 (95% CI 8.2–12.4) months for the sequential and simultaneous groups, respectively [HR 0.83 (95% CI 0.59–1.16); p = 0.274; age-adjusted HR 0.90 (95%CI 0.64–1.27); p = 0.567]. No significant differences were evident for disease-free survival (p = 0.290) or overall survival (p = 0.514) between the two groups. For ER-positive premenopausal patients, the sequential use of GnRHa and chemotherapy showed ovarian preservation and survival outcomes that were no worse than simultaneous use. The application of GnRHa can probably be delayed until menstruation resumption after chemotherapy.

HERV loci transcribed lncRNA TROJAN promotes TNBC progression through ZMYND8 degradation by the ubiquitin-proteasome pathway., Human endogenous retroviruses (HERVs) play pivotal roles in the development of breast cancer. However, the detailed mechanisms of noncoding HERVs remain elusive. Here, our genome-wide transcriptome analysis of HERVs revealed that a primate long noncoding RNA, which we dubbed TROJAN, was highly expressed in human triple-negative breast cancer (TNBC). TROJAN promoted TNBC proliferation and invasion and indicated poor patient outcomes. We further confirmed that TROJAN could bind to ZMYND8, a metastasis-repressing factor, and increase its degradation through the ubiquitin-proteasome pathway by repelling ZNF592. TROJAN also epigenetically up-regulated metastasis-related genes in multiple cell lines. Correlations between TROJAN and ZMYND8 were subsequently confirmed in clinical samples. Furthermore, our study verified that antisense oligonucleotide therapy targeting TROJAN substantially suppressed TNBC progression in vivo. In conclusion, the long noncoding RNA TROJAN promotes TNBC progression and serves as a potential therapeutic target.

Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor 2 (HER2) may be an alternative mechanism to HER2 activation and can affect the sensitivity toward HER2-targeted therapies. We aimed to investigate the prevalence, clinicopathologic characteristics, and functional relevance of novel HER2 mutations in breast cancer. Experimental Design: We performed Sanger sequencing of all exons of the HER2 gene in 1,248 primary tumors and 18 paired metastatic samples. Novel HER2 mutations were functionally characterized. Results: The total HER2 somatic mutation rate was 2.24% (28/1,248). Of the seven novel HER2 mutations, L768S and V773L were only detected in HER2-negative tumors, whereas K753E was found in HER2-positive disease. L768S and V773L mutations exhibited a significant increase in tyrosine kinase–specific activity and strongly increased the phosphorylation of signaling proteins in various cell lines. Xenograft experiments showed that NIH3T3 cells bearing the L768S and V773L mutations displayed more rapid growth. MCF10A, BT474, and MDA-MB-231 cells bearing the K753E mutation were resistant to lapatinib, but could be inhibited by neratinib. Finally, comparison of HER2 mutations in 18 pairs of primary and metastatic lesions revealed that the drug-resistant HER2 mutations (K753E and L755S) were enriched in metastatic lesions. Conclusions: HER2-negative breast cancer with activating mutations can benefit from HER2-targeted therapies. Meanwhile, mutations in the HER2 kinase domain might be a key mechanism of resistance to HER2-targeted therapy, and irreversible tyrosine kinase inhibitors such as neratinib may offer alternative treatment options. Clin Cancer Res; 22(19); 4859–69. ©2016 AACR.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Despite the progress made in precision treatment of cancer patients, targeted treatment is still at its early stage in TNBC, and chemotherapy remains the standard treatment. With the advances in next generation sequencing technology, genomic and transcriptomic analyses have provided deeper insight into the inter-tumoral heterogeneity of TNBC. Much effort has been made to classify TNBCs into different molecular subtypes according to genetic aberrations and expression signatures and to uncover novel treatment targets. In this review, we summarized the current knowledge regarding the molecular classification of TNBC and explore the future paradigm for using molecular classification to guide the development of precision treatment and clinical practice.

We comprehensively analyzed clinical, genomic, and transcriptomic data of a cohort of 465 primary triple-negative breast cancer (TNBC). PIK3CA mutations and copy-number gains of chromosome 22q11 were more frequent in our Chinese cohort than in The Cancer Genome Atlas. We classified TNBCs into four transcriptome-based subtypes: (1) luminal androgen receptor (LAR), (2) immunomodulatory, (3) basal-like immune-suppressed, and (4) mesenchymal-like. Putative therapeutic targets or biomarkers were identified among each subtype. Importantly, the LAR subtype showed more ERBB2 somatic mutations, infrequent mutational signature 3 and frequent CDKN2A loss. The comprehensive profile of TNBCs provided here will serve as a reference to further advance the understanding and precision treatment of TNBC.

Luminal breast cancer (BC) has a sustained risk of late disease recurrence and death. Considerable numbers of patients suffer from antiendocrine therapy resistance. Here, we identified a novel lncRNA whose expression is high in breast cancer and especially higher in luminal breast cancer, dubbed LOL (lncRNA of luminal), that acts as a natural sponge for let-7 microRNAs to regulate tumor growth and tamoxifen resistance. LOL overexpression in parental MCF-7 cells exhibited a proliferative advantage in the addition of tamoxifen than negative control. Knocking down LOL in TamR MCF-7 cells, recovered the sensitivity of cells to tamoxifen. Strikingly, we demonstrated that LOL is transcribed from a genomic locus of an enhancer to maintain its high expression in luminal BC and that it is extremely sensitive to enhancer-regulating factors, such as ZMYND8 and BRD4. Estrogen deprivation or ERα signaling pathway blockage can further stimulate LOL expression, which can promote tumor progression. Clinical analysis of 374 luminal breast cancer samples indicated that LOL is an independent prognostic factor for poor survival in luminal BC. In conclusion, targeting LOL using preclinical/clinical drugs, such as BRD4 inhibitors, may represent a promising approach to inhibit luminal breast cancer progression and tamoxifen resistance.

Purpose Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is primarily treated with trastuzumab and other HER2-targeted therapies. However, approximately 30% of HER2-positive tumors exhibit de novo resistance, whereas 40% acquire resistance to these therapies. The mechanisms underlying drug resistance are unclear, and a better understanding of acquired resistance to HER2-targeted therapy is essential for the development of more effective therapeutics. Methods We enrolled 11 patients with HER2-positive breast cancer, all of whom had been treated with trastuzumab for one year and developed lung metastasis in the follow-up. We conducted exome sequencing of the primary and paired metastatic tumors from four patients. We identified somatic mutations in HER2 (K753E and L755S) affecting the kinase domain only in the metastatic lesions of two cases. Sanger sequencing was performed to analyze all exons of the HER2 gene in the paired primary and metastatic tumors of the remaining seven cases. Furthermore, we enrolled 48 patients with HER2-positive disease who underwent trastuzumab-based neoadjuvant therapy. We performed Sanger sequencing of the HER2 gene in paired tumor DNA before and after neoadjuvant therapy. To determine the phenotype of these mutations, we functionally characterized the HER2 mutations using protein structure analysis, in vitro kinase assays, cell culture, and xenograft experiments. Results We identified somatic mutations in HER2 affecting the kinase domain in four of 11 metastatic tumors (one case with K753E and three cases with L755S). None of the primary tumors harbored HER2 mutations. Furthermore, Sanger sequencing in the neoadjuvant setting revealed two of 48 cases harboring HER2 mutations (K753E and L755S) in the post-treatment samples, none of whom had any HER2 mutation in the paired pre-treatment tumor. Protein structure visualization hinted that the HER2 L753 and L755 side chains were in close proximity to the binding site for small-molecule inhibitors, indicating mutations at this residue might produce drug resistance. In vitro kinase assays revealed that HER2 K753E had greater tyrosine kinase-specific activity than wild-type. HER2 autophosphorylation, phosphorylation of HER2’s dimerization partners (EGFR or HER3) and downstream signaling proteins were significantly upregulated in MCF10A and MCF7 cells overexpressing HER2 K753E or L755S. Trastuzumab or lapatinib treatment did not decrease the number of colonies formed in MCF10A cells transduced with K753E or L755S. MCF7 cells bearing K753E or L755S mutant could reverse the inhibition of lapatinib in xenografts. The MCF10A-HER2 K753E and L755S cells were resistant to trastuzumab (IC50>1000μg/mL) and lapatinib (IC50>10μmol/L) but could be readily inhibited by neratinib (IC50 of 15 nmol/L), an irreversible kinase inhibitor of HER2. Conclusion These data implicate that mutations in HER2 kinase domain are a key mechanism in resistance to HER2-targeted therapy. The selection of functional HER2 mutations might act as potential drivers of trastuzumab resistance during the progression of HER2-positive disease. Furthermore, more potent HER2 inhibitors such as neratinib might be of therapeutic benefit for trastuzumab-resistant breast cancer. Citation Format: Yizhou Jiang, Ke-Da Yu, Wen-Jia Zuo, Yu-Jie Wang, Zhi-Ming Shao. Exome sequencing identified emergence of HER2 kinase domain mutations in trastuzumab-resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-02.

Alternative splicing (AS) and its regulation play critical roles in cancer, yet the dysregulation of AS and its molecular bases in breast cancer development have not yet been elucidated. Using an in vivo CRISPR screen targeting RNA-binding proteins, we identified PHD finger protein 5A (PHF5A) as a key splicing factor involved in tumor progression. PHF5A expression was frequently upregulated in breast cancer and correlated with poor survival, and knockdown of PHF5A significantly suppressed cell proliferation, migration, and tumor formation. PHF5A was required for SF3b spliceosome stability and linked the complex to histones, and the PHF5A–SF3b complex modulated AS changes in apoptotic signaling. In addition, expression of a short truncated FAS-activated serine/threonine kinase (FASTK) protein was increased after PHF5A ablation and facilitated Fas-mediated apoptosis. This PHF5A-modulated FASTK–AS axis was widely present in breast cancer specimens, particularly those of the triple-negative subtype. Taken together, our findings reveal that PHF5A serves as an epigenetic suppressor of apoptosis and thus provides a mechanistic basis for breast cancer progression and may be a valuable therapeutic target. Significance: This study provides an epigenetic mechanistic basis for the aggressive biology of breast cancer and identifies a translatable therapeutic target. Cancer Res; 78(12); 3190–206. ©2018 AACR.

BACKGROUND The GATA3 gene (GATA-binding protein 3) is one of the most frequently mutated genes in breast cancer. The objective of the current study was to determine the clinicopathologic characteristics of patients with breast cancer harboring GATA3 mutations. METHODS The authors examined the somatic mutation status of GATA3 and performed survival analysis in The Cancer Genome Atlas (TCGA) cohort (n = 934) and the Fudan University Shanghai Cancer Center (FUSCC) cohort (n = 308). Patient characteristics, including age; menopausal status; tumor laterality; tumor size; lymph node status; tumor grade; molecular subtypes; adjuvant radiotherapy, chemotherapy, and endocrine therapy; and prognosis, together with PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) and TP53 (tumor protein p53) mutation status, were collected. RESULTS GATA3 mutations were detected in 8.8% of patients (82 of 934 patients) in the TCGA cohort and 14.9% of patients (46 of 308 patients) in the FUSCC cohort. GATA3 mutations were found to be significantly associated with luminal-like breast cancer (P = .002 in the TCGA cohort and P

In this paper, a novel lapping method based on regulating the position of carrier centroid is proposed to modify interfacial normal pressure uniformity. Eight special points are selected to represent carrier weight. This lapping process can be divided into initial stage, regulated stage and stable stage. The purpose of initial stage is calculating the position of carrier centroid according to the equivalent mass of eight points. The regulated stage is to decrease total thickness variation (TTV) by regulating the position of weight. Finally, the stable stage will keep uniformity of material removal rate (MRR) uniform at each point. A 3-inch and 400 μm thickness silicon wafer is lapped to demonstrate the feasibility of this method. We can find that TTV of this wafer decreases from initial stage 20 μm to 3 μm and remain constant. Therefore, the uniformity of MRR has been greatly improved by this novel lapping method.

Purpose Neoadjuvant chemotherapy (NCT) is a standard treatment option for locally advanced breast cancer. However, the lack of an efficient method to predict treatment response and patient prognosis hampers the clinical evaluation of patient eligibility for NCT. An elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with a favorable prognosis for certain hematologic malignancies and for nasopharyngeal carcinoma; however, this association has not been investigated in breast cancer. The purpose of this study was to evaluate whether pre-NCT LMR analysis could predict the prognosis of patients with locally advanced breast cancer. Methods A retrospective cohort of 542 locally advanced breast cancer patients (T3/T4 and/or N2/N3 disease) receiving NCT followed by radical surgery was recruited between May 2002 and August 2011 at the Fudan University Shanghai Cancer Center. Counts for pre-NCT peripheral absolute lymphocytes and monocytes were obtained and used to calculate the LMR. Results Univariate and multivariate analysis revealed that higher LMR levels (≥4.25) were significantly associated with favorable DFS (P = 0.009 and P = 0.011, respectively). Additionally, univariate analysis revealed that a higher lymphocyte count (≥1.5×109/L) showed borderline significance for improved DFS (P = 0.054), while a lower monocyte count (

BACKGROUND: Postmastectomy breast reconstruction is widely used in breast cancer patients for its aesthetic effect. Although several studies have casted suspicion upon the oncological safety of immediate breast reconstruction after mastectomy, the potential impact of different reconstruction methods on patient survival remains unclear. PATIENTS AND METHODS: We identified 35,126 female patients diagnosed with breast cancer from January 1, 1998 to December 31, 2002 in the Surveillance, Epidemiology, and End Results database. Breast cancer-specific survival (BCSS) and overall survival (OS) were compared among patients who underwent mastectomy with or without immediate breast reconstruction (autologous reconstruction or implant reconstruction) using Cox proportional hazard regression models. RESULTS: In multivariate analysis unadjusted for family income, patients undergoing immediate postmastectomy reconstruction exhibited improved BCSS [POOLED reconstruction (any types of reconstruction): hazard ratio (HR) = 0.87, 95% confidence interval (CI) 0.80-0.95, P = 0.001] and OS (pooled reconstruction: HR = 0.70, 95% CI 0.65-0.75, P

The GATA3 gene (GATA-binding protein 3) is one of the most frequently mutated genes in breast cancer. The objective of the current study was to determine the clinicopathologic characteristics of patients with breast cancer harboring GATA3 mutations.The authors examined the somatic mutation status of GATA3 and performed survival analysis in The Cancer Genome Atlas (TCGA) cohort (n=934) and the Fudan University Shanghai Cancer Center (FUSCC) cohort (n=308). Patient characteristics, including age; menopausal status; tumor laterality; tumor size; lymph node status; tumor grade; molecular subtypes; adjuvant radiotherapy, chemotherapy, and endocrine therapy; and prognosis, together with PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) and TP53 (tumor protein p53) mutation status, were collected.GATA3 mutations were detected in 8.8% of patients (82 of 934 patients) in the TCGA cohort and 14.9% of patients (46 of 308 patients) in the FUSCC cohort. GATA3 mutations were found to be significantly associated with luminal-like breast cancer (P=.002 in the TCGA cohort and P.001 in the FUSCC cohort), and were highly mutually exclusive to PIK3CA mutations (P=.001 in the TCGA cohort and P=.003 in the FUSCC cohort) and TP53 mutations (P.001 in both cohorts). Furthermore, GATA3 mutations were correlated with improved overall survival in the entire population (P=.025 in the TCGA cohort and P = .043 in the FUSCC cohort) as well as in patients with luminal-like disease who received adjuvant endocrine therapy.GATA3 mutations mainly occur in patients with luminal-like breast cancer and have identifiable clinicopathologic and genetic characteristics, highlighting a subgroup of patients with breast cancer in whom limited therapy may be appropriate.

Background Triple-negative breast cancer (TNBC) is a highly heterogeneous group of cancers, and molecular subtyping is necessary to better identify molecular-based therapies. While some classifiers have been established, no one has integrated the expression profiles of long noncoding RNAs (lncRNAs) into such subtyping criterions. Considering the emerging important role of lncRNAs in cellular processes, a novel classification integrating transcriptome profiles of both messenger RNA (mRNA) and lncRNA would help us better understand the heterogeneity of TNBC. Methods Using human transcriptome microarrays, we analyzed the transcriptome profiles of 165 TNBC samples. We used k-means clustering and empirical cumulative distribution function to determine optimal number of TNBC subtypes. Gene Ontology (GO) and pathway analyses were applied to determine the main function of the subtype-specific genes and pathways. We conducted co-expression network analyses to identify interactions between mRNAs and lncRNAs. Results All of the 165 TNBC tumors were classified into four distinct clusters, including an immunomodulatory subtype (IM), a luminal androgen receptor subtype (LAR), a mesenchymal-like subtype (MES) and a basal-like and immune suppressed (BLIS) subtype. The IM subtype had high expressions of immune cell signaling and cytokine signaling genes. The LAR subtype was characterized by androgen receptor signaling. The MES subtype was enriched with growth factor signaling pathways. The BLIS subtype was characterized by down-regulation of immune response genes, activation of cell cycle, and DNA repair. Patients in this subtype experienced worse recurrence-free survival than others (log rank test, P = 0.045). Subtype-specific lncRNAs were identified, and their possible biological functions were predicted using co-expression network analyses. Conclusions We developed a novel TNBC classification system integrating the expression profiles of both mRNAs and lncRNAs and determined subtype-specific lncRNAs that are potential biomarkers and targets. If further validated in a larger population, our novel classification system could facilitate patient counseling and individualize treatment of TNBC. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0690-8) contains supplementary material, which is available to authorized users.