FBXL18 is required for ovarian cancer cell proliferation and migration through activating AKT signaling.

BACKGROUND: F-box and leucine-rich repeat protein 18 (FBXL18) is an F-box protein that functions as an E3-ubiquitin ligase, and it plays pivotal roles in multiple disease processes. However, its role and underlying mechanism in ovarian cancer (OC) are still unknown. We investigated the impact and mechanism of FBXL18 in OC cell growth and tumorigenesis. METHODS: Silent interfering RNAs and overexpression plasmids were employed to knock down and overexpress FBXL18 in OC cells (A2780 and OVCAR3). CCK-8, colony formation, cell migration, and nude mouse xenograft assays were used to assess the effect of FBXL18 on OC cell proliferation and migration. Western blotting and co-immunoprecipitation followed by ubiquitination assays were performed to detect the mechanism of the FBXL18/AKT axis in OC. RESULTS: FBXL18 knockdown inhibited OC cell proliferation and migration, whereas FBXL18 overexpression showed the opposite results. Phosphorylated-AKT (S473) protein expression was increased by FBXL18 overexpression and markedly decreased after phosphorylated-AKT inhibitor (MK-2206) treatment. Co-immunoprecipitation assays demonstrated that FBXL18 strongly interacted with AKT in OC cells. Ubiquitination assays revealed that FBXL18 promoted K63-linked AKT ubiquitination to activate AKT. MK-2206 treatment reversed the increase in proliferation and migration of OC cells induced by FBXL18 overexpression. CONCLUSIONS: FBXL18 promoted OC cell proliferation and migration and facilitated OC tumorigenesis. Mechanically, FBXL18 interacted with AKT and promoted K63-linked ubiquitination of AKT to activate AKT in OC cells. Our study revealed that the FBXL18/AKT axis plays a crucial role in the OC process, indicating that FBXL18 may be a valuable target for OC diagnosis and treatment.

Slc26a1 is not essential for spermatogenesis and male fertility in mice.

Thousands of genes are expressed in the testis of mice. However, the details about their roles during spermatogenesis have not been well-clarified for most genes. The purpose of this study was to examine the effect of Slc26a1 deficiency on mouse spermatogenesis and male fertility. Slc26a1-knockout (KO) mice were generated using CRISPR/Cas9 technology on C57BL/6J background. We found no obvious differences between Slc26a1-KO and Slc26a1-WT mice in fertility tests, testicular weight, sperm concentrations, or morphology. Histological analysis found that Slc26a1-KO mouse testes had normal germ cell types and mature sperm. These findings indicated that Slc26a1 was dispensable for male fertility in mice. Our results may save time and resources by allowing other researchers to focus on genes that are more meaningful for fertility studies. We also found that mRNAs of two Slc26a family members (Slc26a5 and Slc26a11) were expressed on higher mean levels in Slc26a1-KO total mouse testes, compared to Slc26a1-WT mice. This effect was not found in mouse GC-1 and GC-2 germ cell lines with the Slc26a1 gene transiently knocked down. This result may indicate that a gene compensation phenomenon was present in the testes of Slc26a1-KO mice.

The effect of sperm DNA fragmentation on in vitro fertilization outcomes of unexplained infertility.

BACKGROUND: Infertility is caused by heterogeneous risks, but most of them are unexplained. The sperm DNA Fragmentation Index (DFI) was increasingly acknowledged as a parameter for the evaluation of male infertility. This study aimed to investigate the association between sperm DFI and laboratory and clinical outcomes in a population with unexplained infertility. METHODS: The clinical data of an infertile population was collected for the selection of reproductive patients with unexplained infertility. The authors classified the patients with normal sperm parameters in a control group (DFI < 25%) and an observation group (DFI ≥ 25%) and compared the difference in basal characteristics, laboratory, and clinical outcomes between the two groups. The authors conducted a correlation analysis to examine the relationship between DFI and the number of D3 good-quality embryos, as well as the clinical pregnancy rate and live birth rate. A total of 176 cases were enrolled in the retrospective study. RESULTS: The observation group (n = 88) showed advanced male age, lower sperm concentration, progressive motility, and morphology assessment than the control group. In addition, lower No. of D3 good-quality embryos, clinical pregnancy rate, and the live birth rate were shown in the observation group. A negative correlation between the DFI and No. of D3 good-quality embryos (rs = -0.347, p < 0.001) or live birth rate (rs = -0.185, p = 0.028) was shown. CONCLUSIONS: Sperm DFI was a good indicator for the prediction of D3 good-quality embryos in unexplained infertility couples, but it did not provide sufficient information regarding clinical pregnancy outcome but live pregnancy outcome.

The effect of sperm DNA fragmentation on in vitro fertilization outcomes of unexplained infertility

Abstract Background Infertility is caused by heterogeneous risks, but most of them are unexplained. The sperm DNA Fragmentation Index (DFI) was increasingly acknowledged as a parameter for the evaluation of male infertility. This study aimed to investigate the association between sperm DFI and laboratory and clinical outcomes in a population with unexplained infertility. Methods The clinical data of an infertile population was collected for the selection of reproductive patients with unexplained infertility. The authors classified the patients with normal sperm parameters in a control group (DFI < 25%) and an observation group (DFI ≥ 25%) and compared the difference in basal characteristics, laboratory, and clinical outcomes between the two groups. The authors conducted a correlation analysis to examine the relationship between DFI and the number of D3 good-quality embryos, as well as the clinical pregnancy rate and live birth rate. A total of 176 cases were enrolled in the retrospective study. Results The observation group (n = 88) showed advanced male age, lower sperm concentration, progressive motility, and morphology assessment than the control group. In addition, lower No. of D3 good-quality embryos, clinical pregnancy rate, and the live birth rate were shown in the observation group. A negative correlation between the DFI and No. of D3 good-quality embryos (rs = -0.347, p < 0.001) or live birth rate (rs = -0.185, p = 0.028) was shown. Conclusions Sperm DFI was a good indicator for the prediction of D3 good-quality embryos in unexplained infertility couples, but it did not provide sufficient information regarding clinical pregnancy outcome but live pregnancy outcome.

Clinical application of artificial neural network (ANN) modeling to predict BRCA1/2 germline deleterious variants in Chinese bilateral primary breast cancer patients.

BACKGROUND: Bilateral breast cancer (BBC), as well as ovarian cancer, are significantly associated with germline deleterious variants in BRCA1/2, while BRCA1/2 germline deleterious variants carriers can exquisitely benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. However, formal genetic testing could not be carried out for all patients due to extensive use of healthcare resources, which in turn results in high medical costs. To date, existing BRCA1/2 deleterious variants prediction models have been developed in women of European or other descent who are quite genetically different from Asian population. Therefore, there is an urgent clinical need for tools to predict the frequency of BRCA1/2 deleterious variants in Asian BBC patients balancing the increased demand for and cost of cancer genetics services. METHODS: The entire coding region of BRCA1/2 was screened for the presence of germline deleterious variants by the next generation sequencing in 123 Chinese BBC patients. Chi-square test, univariate and multivariate logistic regression were used to assess the relationship between BRCA1/2 germline deleterious variants and clinicopathological characteristics. The R software was utilized to develop artificial neural network (ANN) and nomogram modeling for BRCA1/2 germline deleterious variants prediction. RESULTS: Among 123 BBC patients, we identified a total of 20 deleterious variants in BRCA1 (8; 6.5%) and BRCA2 (12; 9.8%). c.5485del in BRCA1 is novel frameshift deleterious variant. Deleterious variants carriers were younger at first diagnosis (P = 0.0003), with longer interval between two tumors (P = 0.015), at least one medullary carcinoma (P = 0.001), and more likely to be hormone receptor negative (P = 0.006) and HER2 negative (P = 0.001). Area under the receiver operating characteristic curve was 0.903 in ANN and 0.828 in nomogram modeling individually (P = 0.02). CONCLUSION: This study shows the spectrum of the BRCA1/2 germline deleterious variants in Chinese BBC patients and indicates that the ANN can accurately predict BRCA deleterious variants than conventional statistical linear approach, which confirms the BRCA1/2 deleterious variants carriers at the lowest costs without adding any additional examinations.

A novel marker based on necroptosis-related long non-coding RNA for forecasting prognostic in patients with clear cell renal cell carcinoma.

Background: The incidence of clear cell renal cell carcinoma (ccRCC) is high and has increased gradually in recent years. At present, due to the lack of effective prognostic indicators, the prognosis of ccRCC patients is greatly affected.Necroptosis is a type of cell death, and along with cell necrosis is considered a new cancer treatment strategy. The aim of this study was to construct a new marker for predicting the prognosis of ccRCC patients based on long non-coding RNA (nrlncRNAs) associated with necroptosis. Methods: RNA sequence data and clinical information of ccRCC patients from the Cancer Genome Atlas database (TCGA) were downloaded. NrlncRNA was identified by Pearson correlation study. The differentially expressed nrlncRNA and nrlncRNA pairs were identified by univariate Cox regression and Lasso-Cox regression. Finally, a Kaplan-Meier survival study, Cox regression, clinicopathological features correlation study, and receiver operating characteristic (ROC) spectrum were used to evaluate the prediction ability of 25-nrlncrnas for markers. In addition, correlations between the risk values and sensitivity to tumor-infiltrating immune cells, immune checkpoint inhibitors, and targeted drugs were also investigated. Results: In the current research, a novel marker of 25-nrlncRNAs pairs was developed to improve prognostic prediction in patients with ccRCC. Compared with clinicopathological features, nrlncRNAs had a higher diagnostic validity for markers, with the 1-year, 3-years, and 5-years operating characteristic regions being 0.902, 0.835, and 0.856, respectively, and compared with the stage of 0.868, an increase of 0.034. Cox regression and stratified survival studies showed that this marker could be an independent predictor of ccRCC patients. In addition, patients with different risk scores had significant differences in tumor-infiltrating immune cells, immune checkpoint, and semi-inhibitory concentration of targeted drugs. The feature could be used to evaluate the clinical efficacy of immunotherapy and targeted drug therapy. Conclusion: 25-nrlncRNAs pair markers may help to evaluate the prognosis and molecular characteristics of ccRCC patients, which improve treatment methods and can be more used in clinical practice.

Drug transport across the blood-testis barrier.

The blood-testis barrier transfers nutrients to spermatogenic tubules to ensure the normal physiological function of the testes. It also restricts the "entry and exit" of biological macromolecules in the testicular lumen and provides a unique microenvironment for spermatogenesis. This makes the testes a safe place for some viruses and tumors, as immune factors cannot function and drugs fail to reach therapeutic concentrations in the testes. This review aimed to describe the factors regulating the structure and physiological function of the blood-testis barrier. By understanding therapeutic mechanisms of action, drugs can be developed to function in the testicles.

Identification of proline-rich protein 11 as a major regulator in mouse spermatogonia maintenance via an increase in BMI1 protein stability.

BACKGROUND: Spermatogenesis accompanied by self-renewal and differentiation of spermatogonia under complicated regulation is crucial for male fertility. Our previous study demonstrated that the loss of the B-lymphoma Mo-MLV insertion region 1 (BMI1) could cause male infertility and found a potential interaction between BMI1 and proline-rich protein 11 (PRR11); however, the specific co-regulatory effects of BMI1/PRR11 on spermatogonia maintenance remain unclear. METHODS AND RESULTS: The expression of PRR11 was downregulated in a mouse spermatogonia cell line (GC-1) via transfection with PRR11-siRNAs, and PRR11 knockdown was verified by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). The proliferative activity of GC-1 cells was determined using the cell counting kit (CCK-8), colony formation, and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. A Transwell assay was performed to evaluate the effects of PRR11 on GC-1 cell migration. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to measure GC-1 cell apoptosis. Furthermore, co-immunoprecipitation, RT-qPCR, and western blot analyses were used for investigating the regulatory mechanisms involved in this regulation. It was found that downregulation of PRR11 could cause a marked inhibition of proliferation and migration and induced apoptosis in GC-1 cells. Moreover, silencing of PRR11 obviously led to a reduction in the BMI1 protein level. PRR11 was found to interact with BMII at the endogenous protein level. PRR11 knockdown produced a decrease in BMI1 protein stability via an increase in BMI1 ubiquitination after which derepression in the transcription of protein tyrosine phosphatase receptor type M (Ptprm) occurred. Importantly, knockdown of Ptprm in PRR11-deficient GC-1 cells led to a reversal of proliferation and migration of GC-1 cells. CONCLUSIONS: This study uncovered a novel mechanism by which PRR11 cooperated with BMI1 to facilitate GC-1 maintenance through targeting Ptprm. Our findings may provide a better understanding of the regulatory network in spermatogonia maintenance.

SAT2 regulates Sertoli cell-germline interactions via STIM1-mediated ROS/WNT/ß-catenin signaling pathway.

As the main component of seminiferous tubules, Sertoli cells are in close contact with germ cells and generate niche signals, which exhibit pivotal functions in spermatogenesis and male fertility. However, the regulatory mechanisms of Sertoli cell-germline interactions (SGIs) in the testes of neonatal mice (NM) remain largely unclear. Previously, we identified spermidine/spermine N1-acetyl transferase 2 (SAT2) and stromal interaction molecule 1 (STIM1) to be potential regulators of testicular cord formation via comparative proteomics analysis. Here, we demonstrated a novel role of SAT2 for SGIs during testicular development in NM. Testicular explants lacking SAT2 affected the mislocation, but not the quantity, of Sertoli cells, which led to maintenance defects in spermatogonial stem cells (SSCs). Interestingly, SAT2 was essential for the migration of TM4 cells, a Sertoli cell line. Mechanistically, SAT2 was able to bind STIM1, repress its expression, and regulate homeostasis of a reactive oxygen species/wingless type (WNT)/ß-catenin pathway in NM testes. Collectively, our study identified that SAT2 was able to regulate SGIs via a STIM1-mediated WNT signaling pathway.

Quantitative proteomics and functional analysis identified novel targets for missed abortion.

Missed abortion (MA) is a special form of spontaneous abortion that is increasing in incidence. However, the precise molecular mechanisms underlying MA, especially regarding the decidua, are poorly understood. Herein, we identified molecular signaling pathways related to MA by comparing the decidua of women experiencing normal pregnancy and MA using a quantitative proteomics approach based on HPLC-MS/MS and iTRAQ labeling. Integrated bioinformatics analysis of villi and decidua was performed to reveal potential crosstalk signals in closely related tissues. We identified 2277 proteins with high confidence in decidua, of which 232 were differentially expressed in MA samples. Specifically, we reported that integrated quantitative proteomic and bioinformatic analysis revealed altered proteins in MA and the mechanisms underpinning MA involved numerous pathways, especially ribosome and cellular metabolism signaling. Moreover, Importin 9, Cullin 1 and COX6C are critical for MA, and their altered expression might contribute to the pathophysiology of MA. In particular, COX6C was dramatically down-regulated in both decidua and villi of MA. COX6C was also found to be highly expressed in syncytiotrophoblastic and cytotrophoblastic cells in villi and widely expressed in decidua of the control group, but dramatically decreased in the MA group. Functional analysis showed that knockdown of COX6C inhibited apoptosis process in both HTR-8 and SiHa cells, suggesting that COX6C may play protective effects in MA. Thus, this study could help to map the regulatory protein network related to MA and contribute to the pathophysiological mechanisms of MA.

BMI1 promotes spermatogonial stem cell maintenance by epigenetically repressing Wnt10b/ß-catenin signaling.

The self-renewal of spermatogonial stem cells (SSCs) requires a special microenvironment and is strictly controlled. Previously, we identified BMI1 as a key regulator of spermatogenesis in a knock-out mouse model. However, the mechanisms by which BMI1 regulates SSC maintenance remain largely unknown. Herein, we show that BMI1 is essential for SSC maintenance. BMI1 directs the transcriptional repression of target genes by increasing H2AK119ub and reducing H3K4me3 in SSCs. Furthermore, BMI1 inhibition resulted in the transcriptional activation of Wnt10b and thereby promoted the nuclear translocation of ß-catenin in SSCs. Importantly, the suppression of Wnt/ß-catenin signaling restored both the cytoplasmic expression of ß-catenin and SSC maintenance in BMI1-deficient SSCs. Finally, we demonstrated that Wnt/ß-catenin signaling was also involved in BMI1-mediated SSC maintenance in vivo. Altogether, our study not only reveals a novel mechanism for BMI1 in the process of SSC maintenance, but also provides a potential new strategy for treating male infertility.

Testis-enriched Asb12 is not required for spermatogenesis and fertility in mice.

Background: Members of the ankyrin repeat and SOCS box (Asb) family are expressed abundantly in testes. Some Asb genes/proteins are required for spermatogenesis, but the function of Asb12 during spermatogenesis is not clear. We investigated the physiological role of Asb12 in murine testes. Methods: The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system was used to generate Asb12-knockout (KO) mice. Histology and immunostaining were done to assess the effects of Asb12 KO on mouse testes and epididymides. Semen quality was analyzed using a computer-assisted sperm analyzer. The terminal deoxynucleotidyl transferase-dUTP nick-end labeling assay was employed to examine testicular apoptosis. Real-time reverse transcription-quantitative polymerase chain reaction (PCR) was conducted to calculate gene transcription levels. Results: Asb12 was expressed predominantly in murine testes. Immunostaining of Asb12 protein revealed that Asb12 was located specifically in the acrosome of elongated spermatids, which suggested a potential role of Asb12 during spermatogenesis. However, Asb12-KO mice had normal fertility, and no overt difference was detected in testicular morphology, semen quality, or apoptosis when comparing Asb12-KO and Asb12-wild type (WT) mice. Gene expression of several Asb family members was increased significantly in the testes of Asb12-KO mice when compared with that in Asb12-WT mice, which suggested functional compensation from paralogs for Asb12 loss. Conclusions: We demonstrated that Asb12 is not essential for the spermatogenesis and fertility of mice. Our findings will assist researchers in avoiding redundant efforts, and provide a baseline resource for genetic studies on human fertility.

LINC00624/TEX10/NF-κB axis promotes proliferation and migration of human prostate cancer cells.

Prostate cancer (PCa) is a malignant epithelial tumor with a high rate of biochemical or local recurrence. Studies have suggested that LINC00624 plays an important oncogenic role in liver cancer. However, whether it exerts similar effects in PCa progression remains unknown. In this study, we explored the effects of LINC00624 on the malignant progression of PCa and sought to identify the relevant signaling pathways. The results showed that LINC00624 was highly expressed in PCa tissues and cells and was associated with poor prognosis in PCa patients. In vitro and in vivo assays further showed that LINC00624 knockdown could decrease the proliferative and migratory ability of PCa cells. Mechanistically, we found that LINC00624 and TEX10 formed a co-regulatory axis that stimulated NF-κB activity. Our data suggest that LINC00624 acts as an oncogene in PCa progression and has potential as a novel biomarker for PCa.

Identification of bi-allelic KIF9 loss-of-function variants contributing to asthenospermia and male infertility in two Chinese families.

Introduction: Asthenozoospermia (AZS) is a leading cause of male infertility, affecting an estimated 18% of infertile patients. Kinesin proteins function as molecular motors capable of moving along microtubules. The highly conserved kinesin family member 9 (KIF9) localizes to the central microtubule pair in the flagella of Chlamydomonas cells. The loss of KIF9 expression in mice has been linked to AZS phenotypes. Methods: Variant screening was performed by whole exome sequencing from 92 Chinese infertile patients with AZS. Western blot was used to was used for analyzing of candidate proteins expression. Patients' sperm samples were stained with immunofluorescent to visualise proteins localization and were visualised by transmission electron microscopy (TEM) to determine axoneme structures. Co-immunoprecipitation assay was used to verify the binding proteins of KIF9. In vitro fertilization (IVF) was used to evaluate the efficiency of clinical treatment. Results: Bi-allelic KIF9 loss-of-function variants were identified in two unrelated Chinese males exhibiting atypical sperm motility phenotypes. Both of these men exhibited typical AZS and suffered from infertility together with the complete absence of KIF9 expression. In contrast to these KIF9-deficient patients, positive KIF9 staining was evident throughout the flagella of sperm from normal control individuals. KIF9 was able to interact with the microtubule central pair (CP) component hydrocephalus-inducing protein homolog (HYDIN) in human samples. And KIF9 was undetectable in spermatozoa harboring CP deletions. The morphologicy of KIF9-deficient spermatozoa appeared normal under gross examination and TEM. Like in mice, in vitro fertilization was sufficient to overcome the fertility issues for these two patients. Discussion: These findings indicate that KIF9 associates with the central microtubules in human sperm and that it functions to specifically regulate flagellar swinging. Overall, these results offer greater insight into the biological functions of KIF9 in the assembly of the human flagella and its role in male fertility.

MRNIP is essential for meiotic progression and spermatogenesis in mice.

Meiotic homologous recombination (HR) initiates with the programmed generation of DNA double-strand breaks (DSBs), which result in the exchange of genetic information and genome diversity. This process requires the tight cooperation of the MRE11-RAD50-NBS1 (MRN) complex to promote DSB formation and DNA end resection. However, the mechanism regulating MRN complex remains to be explored. In the present study, we report that MRN-interacting protein, MRNIP, is a novel factor for HR and is crucial for the expression of the MRN complex and loading of recombinases DMC1/RAD51. Knockout of Mrnip in mice led to aberrant synapsis, impaired HR, and male subfertility. In conclusion, MRNIP is a novel HR factor that probably promotes meiotic progression through the MRN complex.

E3 ubiquitin ligase ASB17 is required for spermiation in mice.

BACKGROUND: A major goal of spermiation is to degrade the apical ectoplasmic specialization (ES) junction between Sertoli cells and elongating spermatids in preparation for the eventual disengagement of spermatids into the lumen. E3 ubiquitin ligases mediate the process of ubiquitination and the subsequent proteasomal degradation, but their specific role during spermiation remains largely unexplored. METHODS: Ankyrin repeat and SOCS box protein 17 (Asb17)-knockout mice were generated via a CRISPR/Cas9 approach. Epididymal sperm parameters were assessed by a computer-assisted sperm analysis (CASA) system, and morphological analysis of testicular tissues were performed based on histological and immunostaining staining, and transmission electron microscopy (TEM). The interactions between ASB17 and Espin (ESPN) were predicted by HawkDock server and validated through protein pull-down and immunoprecipitation assays. RESULTS: We report that ASB17, an E3 ligase, is required for the completion of spermiation and that mice lacking Asb17 are oligozoospermic owing to spermiation failure. ASB17-deficient mice are fertile; however, spermatids exhibit a disorganized ES junction, resulting in retention within the seminiferous epithelium. Mechanistically, ASB17 deficiency leads to excess accumulation of ESPN, an actin-binding essential structural component of the ES. We determined that ASB17 regulates the removal of the ES through ubiquitin mediated protein degradation of ESPN. CONCLUSIONS: In summary, our study describes a role for ASB17 in the regulation of cell-cell junctions between germ cells and somatic cells in the testis. These findings establish a novel mechanism for the regulatory role of E3 ligases during spermatogenesis.

Transcriptional Expressions of CXCL9/10/12/13 as Prognosis Factors in Breast Cancer.

CXCLs play critical roles in antitumor immunity by activating tumor-specific immune responses and stimulating tumor proliferation, thus affecting patient outcomes. However, the expression and prognostic values of CXCLs in breast cancer have not been well clarified. The aim of this study was to investigate the impact of CXCLs transcriptional expression on breast cancer patients. Oncomine database, GEPIA (Gene Expression Profiling Interactive Analysis), UALCAN, Kaplan-Meier Plotter, TIMER (Tumor Immune Estimation Resource), and DAVID were used in our study. The transcriptional levels of CXCL9/10/11/13 in breast cancer tissues were significantly elevated while the transcriptional levels of CXCL1/2/3/12 were decreased based on intersections of Oncomine database and GEPIA. Among them, breast cancer patients with high transcriptional levels of CXCL2/9/10/12/13 and low transcriptional level of CXCL3 were associated with a better prognosis. We also found that most of CXCLs expressions are significantly correlated with known prognostic factors, such as patient's age, major subclasses, individual cancer stages, and nodal metastasis status. In addition, the expression of CXCL9/10/12/13 was also indicated to be correlated with the infiltration of six types of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). The functions of differentially expressed CXCLs are primarily related to the immune response and cytokine-cytokine receptor interactions. Our results may provide novel evidence of new prognostic or predictive biomarkers for breast cancer patients.

Dual HER2 Blockade versus a Single Agent in Trastuzumab-Containing Regimens for HER2-Positive Early Breast Cancer: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

PURPOSE: Although trastuzumab is the standard of care for patients with human epidermal growth factor receptor 2 (HER2)- positive early breast cancer (EBC), drug resistance and disease relapse occur. Therefore, we performed a meta-analysis to assess the efficacy and safety of trastuzumab-containing dual anti-HER2 therapy compared to trastuzumab alone. METHODS: A systematic search was performed to identify eligible randomized controlled trials (RCTs). Main outcomes including event-free survival/invasive disease-free survival (EFS/iDFS), overall survival (OS), and safety were considered. RESULTS: Ten RCTs were included (15,284 patients). Significant improvements were observed in both EFS/iDFS (HR 0.86, p=0.0003) and OS (HR 0.86, p=0.02) with trastuzumab-based dual anti-HER2 therapy, especially in adjuvant treatment, while in the neoadjuvant setting, dual-targeted therapy also achieved a substantial pathological complete response (pCR) benefit (HR 1.34, p=0.0002). Subgroup analysis revealed that the EFS/iDFS benefit was slightly higher with trastuzumab plus pertuzumab or plus neratinib than trastuzumab plus lapatinib, while OS benefit was significant with trastuzumab plus lapatinib, but there were no subgroup differences (interaction test, p=0.80 and 0.24, resp.). In addition, EFS/iDFS benefit was unrelated to hormone receptor status but pronounced in the lymph node-positive (LN+) subgroup, which should be interpreted cautiously for lacking interaction (p=0.18). Besides, patients receiving dual therapy, especially with the lapatinib-containing regimen, experienced more toxicity, but no increase in cardiotoxicity. CONCLUSIONS: Despite being associated with more toxicity, trastuzumab-containing dual anti-HER2 therapy is superior to trastuzumab single agent for HER2-positive EBC independent of hormone receptor status. The correlation between survival and LN status needs further verification. Trastuzumab plus pertuzumab or plus neratinib is the preferred regimen with substantial efficacy and lower toxicity.