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Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process.In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 μg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR.As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR.Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Antibiotic resistance is the principal mechanism of an evergrowing bacterial threat. Antibiotic residues in the environment are a major contributor to the spread of antibiotic resistance genes (ARGs). Subinhibitory concentrations of antibiotics cause bacteria to produce reactive oxygen species (ROS), which can lead to mutagenesis and horizontal gene transfer (HGT) of ARGs; however, little is known about the mitigation of ARG dissemination through ROS removal by antioxidants. In this study, we examine how antioxidant-producing microorganisms inoculated in replicate activated sludge systems can biologically mitigate the dissemination of ARGs. Through quantitative polymerase chain reaction (qPCR), we showed that antioxidant-producing microorganisms could decrease the persistence of the RP4 plasmid and alleviate enrichment of ARGs (sul1) and class 1 integrons (intl1). Metagenomic sequencing identified the most diverse resistome and the most mutated Escherichia coli ARGs in the reactor that contained antibiotics but no antioxidant-producing microorganisms, suggesting that antioxidant-producing microorganisms mitigated ARG enrichment and mutation. Host classification revealed that antioxidant-producing microorganisms decreased the diversity of ARG hosts by shaping the microbial community through competition and functional pathway changes. Conjugative experiments demonstrated that conjugative transfer of ARGs could be mitigated by coculture with antioxidant-producing microorganisms. Overall, this is a novel study that shows how ARG enrichment and HGT can be mitigated through bioaugmentation with antioxidant-producing microorganisms.

Objectives This study aimed to investigate whether plasma levels of adenosine, adenosine deaminase (ADA), and certain cytokines change in patients with chronic insomnia disorder (CID), and if so, whether these alterations are associated with poor sleep quality and cognitive dysfunction. Methods Fifty-five CID patients were selected for the study, along with fifty-five healthy controls (HC) matched to the patients according to their basic data. All subjects completed sleep, emotion, and cognition assessments, with some CID patients also completing an overnight polysomnography. The plasma level of adenosine was measured using liquid chromatography–tandem mass spectrometry, while ADA level was quantified using a quantitative sandwich enzyme-linked immunosorbent assay. Levels of cytokines, including IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α, and IFN-γ, were measured using Luminex liquid chip technology. Results CID patients had a lower adenosine level, and higher levels of ADA and some of the cytokines (IL-1β, IL-2, IL-6, IL-10 and TNF-α) compared with controls. In the CID group, plasma concentrations of adenosine were negatively correlated with Pittsburgh Sleep Quality Index scores, while concentrations of IL-1β, IL-6 and TNF-α were positively correlated with these scores. Concentrations of IL-1β and TNF-α were negatively correlated with scores on the Chinese-Beijing Version of the Montreal Cognitive Assessment. Moreover, levels of IL-1β, TNF-α, IL-6, and IL-2 were positively correlated with memory test errors by CID patients after controlling for confounding factors. Conclusions The reduced adenosine and elevated cytokine levels of CID patients were associated with the severity of insomnia and/or cognitive dysfunction.

Ya-Hong Zheng,1,* Chong-Yang Ren,2,* Ying Shen,3 Jia-Bin Li,1,4– 6 Ming-Wei Chen7 1Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China; 2Department of Neurology (Sleep Disorder), The Affiliated Chaohu Hospital of Anhui Medical University, Chaohu 238000, People’s Republic of China; 3Department of Endocrinology, Nanjing Tongren Hospital, Nanjing 211102, People’s Republic of China; 4Department of Infectious Diseases, The Affiliated Chaohu Hospital of Anhui Medical University, Chaohu 238000, People’s Republic of China; 5Anhui Center for Surveillance of Bacterial Resistance, Hefei 230022, People’s Republic of China; 6Institute of Bacterial Resistance, Anhui Medical University, Hefei 230022, People’s Republic of China; 7Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ming-Wei ChenDepartment of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of ChinaEmail chmw1@163.comJia-Bin LiDepartment of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of ChinaEmail lijiabin@ahmu.edu.cnObjective: This study explored the changes in the levels of IL-6, IL-17, TNF-α, and TNF-β, whether such changes were associated with anxiety and depression in diabetic peripheral neuropathy (DPN), and what factors associated with the occurrence of DPN.Methods: Forty-four patients diagnosed with DPN comprised the DPN group, including DPN1 (mild diabetic peripheral neuropathy, 29 cases) and DPN2 groups (moderate-severe diabetic peripheral neuropathy, 15 cases). Thirty-seven individuals with type 2 diabetes mellitus constituted the diabetes mellitus with no neuropathy (NDPN) group. Electromyography was applied to confirm DPN, and the Toronto clinical scoring system (TCSS) score was used to assess the severity of DPN. All subjects’ emotions were evaluated using the self-rating anxiety scale (SAS) and self-rating depression scale (SDS). Triiodothyronine (T3), tetraiodothyronine (T4), and thyroid-stimulating hormone (TSH) levels were measured using chemiluminescent immunoassay. The relevant biochemical indicators were detected using an automatic biochemical analyzer. The plasma levels of cytokines were detected using quantitative sandwich enzyme-linked immunosorbent assay.Results: Patients with DPN had elevated levels of anxiety, IL-6, IL-17, and TNF-α. There were some positive associations between negative emotions and cytokines. The TCSS score positively correlated with IL-17, SAS score, and T3. DPN independently correlated with age, disease duration, fasting plasma glucose (FPG), and IL-17. The combination of IL-17 and TNF-α had higher diagnostic value for DPN than any single cytokine.Conclusion: Patients with DPN had elevated levels of inflammatory cytokines, which were associated with negative emotion, and IL-17 had independent correlation with DPN.Keywords: diabetic peripheral neuropathy, emotions, cytokines

Increasing evidence indicates that exposure to inflammation during pregnancy intensifies the offspring’s cognitive impairment during aging, which might be correlated with changes in some synaptic plasticity-related proteins. In addition, an enriched environment (EE) can significantly exert a beneficial impact on cognition and synaptic plasticity. However, it is unclear whether gestational inflammation combined with postnatal EE affects the changes in cognition and synaptic plasticity-related proteins during aging. In this study, pregnant mice were intraperitoneally injected with lipopolysaccharides (LPS, 50 μg/kg) or normal saline at days 15–17 of pregnancy. At 21 days after delivery, some LPS-treated mice were randomly selected for EE treatment. At the age of 6 and 18 months, Morris water maze (MWM) and western blotting were, respectively, used to evaluate or measure the ability of spatial learning and memory and the levels of postsynaptic plasticity-related proteins in the hippocampus, including postsynaptic density protein 95 (PSD-95), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluA1 subunit, and Homer-1b/c. The results showed that 18-month-old control mice had worse spatial learning and memory and lower levels of these synaptic plasticity-related proteins (PSD-95, GluA1, and Homer-1b/c) than the 6-month-old controls. Gestational LPS exposure exacerbated these age-related changes of cognition and synaptic proteins, but EE could alleviate the treatment effect of LPS. In addition, the performance during learning and memory periods in the MWM correlated with the hippocampal levels of PSD-95, GluA1, and Homer-1b/c. Our results suggested that gestational inflammation accelerated age-related cognitive impairment and the decline of PSD-95, GluA1, and Homer-1b/c protein expression, and postpartum EE could alleviate these changes.

BackgroundTriple-negative breast cancer (TNBC) is refractory and heterogeneous, comprising various entities with divergent phenotype, biology, and clinical presentation. As an aggressive subtype, Chinese TNBC patients with special morphologic patterns (STs) were restricted to its incidence of 10-15% in total TNBC population.MethodsWe recruited 89 patients with TNBC at Guangdong Provincial People’s Hospital (GDPH) from October 2014 to May 2021, comprising 72 cases of invasive ductal carcinoma of no-special type (NSTs) and 17 cases of STs. The clinical data of these patients was collected and statistically analyzed. Formalin-fixed, paraffin-embedded (FFPE) tumor tissues and matched blood samples were collected for targeted next-generation sequencing (NGS) with cancer-related, 520- or 33-gene assay. Immunohistochemical analysis of FFPE tissue sections was performed using anti-programmed cell death-ligand 1(PD-L1) and anti-androgen receptor antibodies.ResultsCases with NSTs presented with higher histologic grade and Ki-67 index rate than ST patients (NSTs to STs: grade I/II/III 1.4%, 16.7%,81.9% vs 0%, 29.4%, 58.8%; pTP53 (88.7%), PIK3CA (26.8%), MYC (18.3%) in NSTs, and TP53 (68.8%), PIK3CA (50%), JAK3 (18.8%), KMT2C (18.8%) in STs respectively. Compared with NSTs, PIK3CA and TP53 mutation frequency showed difference in STs (47.1% vs 19.4%, p=0.039; 64.7% vs 87.5%, p=0.035).ConclusionsIn TNBC patients with STs, decrease in histologic grade and ki-67 index, as well as increase in PD-L1 and AR expression were observed when compared to those with NSTs, suggesting that TNBC patients with STs may better benefit from immune checkpoint inhibitors and/or AR inhibitors. Additionally, lower TP53 and higher PIK3CA mutation rates were also found in STs patients, providing genetic evidence for deciphering at least partly potential mechanism of action.

Natural polyketides play important roles in clinical treatment, agriculture, and animal husbandry. Compared to natural hosts, heterologous chassis (especially Actinomycetes) have many advantages in production of polyketide compounds. As a widely studied model Actinomycete, Saccharopolyspora erythraea is an excellent host to produce valuable heterologous polyketide compounds. However, many host factors affect the expression efficiency of heterologous genes, and it is necessary to modify the host to adapt heterologous production. In this study, the CRISPR-Cas9 system was used to knock out the erythromycin biosynthesis gene cluster of Ab (erythromycin high producing stain). A fragment of 49491 bp in genome (from SACE_0715 to SACE_0733) was deleted, generating the recombinant strain AbΔery in which erythromycin synthesis was blocked and synthetic substrates methylmalonyl-CoA and propionyl-CoA accumulated enormously. Based on AbΔery as heterologous host, three genes, AsCHS, RgTAL, and Sc4CL, driven by strong promoters Pj23119, PermE, and PkasO, respectively, were introduced to produce novel polyketide by L-tyrosine and methylmalonyl-CoA. The product (E)-4-hydroxy-6-(4-hydroxystyryl)-3,5-dimethyl-2H-pyrone was identified in fermentation by LC-MS. High performance liquid chromatography analysis showed that knocking out ery BGC resulted in an increase of methylmalonyl-CoA by 142% and propionyl-CoA by 57.9% in AbΔery compared to WT, and the yield of heterologous product in AbΔery:AsCHS-RgTAL-Sc4CL was higher than WT:AsCHS-RgTAL-Sc4CL. In summary, this study showed that AbΔery could potentially serve as a precious heterologous host to boost the synthesis of other valuable polyketone compounds using methylmalonyl-CoA and propionyl-CoA in the future.

Accumulating evidence has indicated that embryonic inflammation could accelerate age-associated cognitive impairment, which can be attributed to dysregulation of synaptic plasticity-associated proteins, such as RNA-binding proteins (RBPs). Staufen is a double-stranded RBP that plays a critical role in the modulation of synaptic plasticity and memory. However, relatively few studies have investigated how embryonic inflammation affects cognition and neurobiology during aging, or how the adolescent psychosocial environment affects inflammation-induced remote cognitive impairment. Consequently, the aim of this study was to investigate whether these adverse factors can induce changes in Staufen expression, and whether these changes are correlated with cognitive impairment. In our study, CD-1 mice were administered lipopolysaccharides (LPS, 50 μg/kg) or an equal amount of saline (control) intraperitoneally during days 15-17 of gestation. At 2 months of age, male offspring were randomly exposed to stress (S), an enriched environment (E), or not treated (CON) and then assigned to five groups: LPS, LPS+S, LPS+E, CON, and CON+S. Mice were evaluated at 3-month-old (young) and 15-month-old (middle-aged). Cognitive function was assessed using the Morris water maze test, while Staufen expression was examined at both the protein and mRNA level using immunohistochemistry/western blotting and RNAscope technology, respectively. The results showed that the middle-aged mice had worse cognitive performance and higher Staufen expression than young mice. Embryonic inflammation induced cognitive impairment and increased Staufen expression in the middle-aged mice, whereas adolescent stress/an enriched environment would accelerated/mitigated these effects. Meanwhile, Staufen expression was closely correlated with cognitive performance. Our findings suggested embryonic inflammation can accelerate age-associated learning and memory impairments, and these effects may be related to the Staufen expression.

Background Cyanobacteria are of special concern because they proliferate in eutrophic water bodies worldwide and affect water quality. As an ancient photosynthetic microorganism, cyanobacteria can survive in ecologically diverse habitats because of their capacity to rapidly respond to environmental changes through a web of complex signaling networks, including using second messengers to regulate physiology or metabolism. A ubiquitous second messenger, bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has been found to regulate essential behaviors in a few cyanobacteria but not Microcystis, which are the most dominant species in cyanobacterial blooms. In this study, comparative genomics analysis was performed to explore the genomic basis of c-di-GMP signaling in Microcystis aeruginosa. Results Proteins involved in c-di-GMP metabolism and regulation, such as diguanylate cyclases, phosphodiesterases, and PilZ-containing proteins, were encoded in M. aeruginosa genomes. However, the number of identified protein domains involved in c-di-GMP signaling was not proportional to the size of M. aeruginosa genomes (4.97 Mb in average). Pan-genome analysis showed that genes involved in c-di-GMP metabolism and regulation are conservative in M. aeruginosa strains. Phylogenetic analysis showed good congruence between the two types of phylogenetic trees based on 31 highly conserved protein-coding genes and sensor domain-coding genes. Propensity for gene loss analysis revealed that most of genes involved in c-di-GMP signaling are stable in M. aeruginosa strains. Moreover, bioinformatics and structure analysis of c-di-GMP signal-related GGDEF and EAL domains revealed that they all possess essential conserved amino acid residues that bind the substrate. In addition, it was also found that all selected M. aeruginosa genomes encode PilZ domain containing proteins. Conclusions Comparative genomics analysis of c-di-GMP metabolism and regulation in M. aeruginosa strains helped elucidating the genetic basis of c-di-GMP signaling pathways in M. aeruginosa. Knowledge of c-di-GMP metabolism and relevant signal regulatory processes in cyanobacteria can enhance our understanding of their adaptability to various environments and bloom-forming mechanism.

Horizontal transfer of catabolic plasmids is used in genetic bioaugmentation for environmental pollutant remediation. In this study, we examined the effectiveness of genetic bioaugmentation with dioxin-catabolic plasmids harbored by Rhodococcus sp. strain p52 in laboratory-scale sequencing batch reactors (SBRs). During 100 days of operation, bioaugmentation decreased the dibenzofuran content (120 mg L–1) in the synthetic wastewater by 32.6%–100% of that in the nonbioaugmented SBR. Additionally, dibenzofuran was removed to undetectable levels in the bioaugmented SBR, in contrast, 46.8 ± 4.1% of that in the influent remained in the nonbioaugmented SBR after 96 days. Moreover, transconjugants harboring pDF01 and pDF02 were isolated from the bioaugmented SBR after 2 days, and their abilities to degrade dibenzofuran were confirmed. After 80 days, the copy numbers of strain p52 decreased by 3 orders of magnitude and accounted for 0.05 ± 0.01% of the total bacteria, while transconjugants were present at around 1...

Cyanobacterial blooms can cause serious environmental problems and threaten aquatic organisms and human health. It is therefore essential to effectively control cyanobacterial blooms in aquatic ecosystems. In the present study, the anticyanobacterial effect of L-lysine on Microcystis aeruginosa was examined. The results showed that the growth of M. aeruginosa (>90%) was effectively inhibited by L-lysine at dosages of 5.0, 6.5, and 8.0 mg L−1 after 3 d treatment. The content of superoxide anion radicals, MDA content and SOD activity in M. aeruginosa cells increased after 1 d of treatment with L-lysine (3.0, 5.0, 6.5, and 8.0 mg L−1), revealing that L-lysine induced oxidative stress in the cyanobacterial cells. The chlorophyll-a and protein contents in M. aeruginosa treated with L-lysine (3.0, 5.0, 6.5, and 8.0 mg L−1) decreased after 2 d, indicating damage of the photosynthetic system by L-lysine treatment. Additionally, the production of exopolysaccharide by M. aeruginosa also increased and the expression of polysaccharide synthesis genes was upregulated by 3.0 mg L−1 L-lysine after 3 d of treatment. In response to the algicidal effects of L-Lysine, M. aeruginosa upregulated exopolysaccharide synthesis. Electron microscopic observations demonstrated that the cell membrane of M. aeruginosa was broken down during treatment with L-lysine (≥3.0 mg L−1). Our results revealed that the effects of L-lysine on M. aeruginosa cells were comprehensive, and L-lysine is therefore an efficient anticyanobacterial reagent.

Objective Although circulating tumor cells (CTCs) have been well-established as promising prognostic biomarkers in both early breast cancer and metastatic settings, little is known regarding the prognostic relevance of CTCs in the long-term postoperative monitoring of patients with non-metastatic breast cancer (non-MBC). In this study, we investigated the associations of CTCs with clinicopathological features and metabolic-related variables, such as obesity and hyperglycemia. Methods In this retrospective study, we recruited 264 patients with postoperative stage I-III breast cancer at Guangdong General Hospital from January 2009 to December 2015. The prevalence and number of CTCs were assessed using the CellSearch System at a median time of 19.0 months [interquartile range (IQR), 7.8-33.0] after surgery. The CTC assay results were correlated with the clinicopathological features and metabolic-related variables. A multivariate logistic regression analysis was performed to further determine the independent predictors of CTCs. Results CTCs were detected in 10.6% of all patients. The positive rate of CTCs in patients with infiltrating ductal carcinoma was lower than that in patients with other pathological types (9.0% vs. 28.6%, P=0.020). More importantly, the presence of CTCs was correlated with blood glucose level (P=0.015) and high-density lipoprotein level (P=0.030). The multivariate logistic regression analysis showed that the pathological type [odds ratio (OR): 1.757, 95% CI: 1.021-3.023; P=0.042] and blood glucose level (OR: 1.218, 95% CI: 1.014-1.465; P=0.035) were independent predictors of the presence of CTCs. Conclusions This study revealed potential associations between CTCs and metabolic-related factors in Chinese patients with non-MBC and supports the hypothesis that metabolic dysfunction in breast cancer patients might influence the biological activity of metastatic breast cancer, leading to a higher prevalence of CTCs.

The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 ( eryBIV)-SACE_0721 ( eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.

Microcystis, the dominant species among cyanobacterial blooms, normally forms colonies under natural conditions but exists as single cells or paired cells in axenic laboratory cultures after long-term cultivation. Here, a bloom-forming Microcystis aeruginosa strain CHAOHU 1326 was studied because it presents a colonial morphology and grows on the water surface during axenic laboratory culturing. We first examined the morphological features of strain CHAOHU 1326 and three other unicellular M. aeruginosa strains FACHB-925, FACHB-940, and FACHB-975 cultured under the same conditions by scanning and transmission electron microscopy. Then, we compared the extracellular polysaccharide (EPS)-producing ability of colonial strain CHAOHU 1326 to that of the three unicellular M. aeruginosa strains, and found that strain CHAOHU 1326 produced a higher amount of EPS than the other strains during growth. Moreover, based on genome sequencing, multiple gene clusters implicated in EPS biosynthesis and a cluster of 12 genes predicted to be involved in gas vesicle synthesis in strain CHAOHU 1326 were detected. These predicted genes were all functional and expressed in M. aeruginosa CHAOHU 1326 as determined by reverse transcription PCR. These findings provide a physiological and genetic basis to better understand colony formation and buoyancy control during M. aeruginosa blooming.

552 Background: Extensive efforts by The Cancer Genome Atlas (TCGA) Network had provided much of our current understanding of the molecular profile of various solid tumors including breast tumors; however, Asian patients were underrepresented in this cohort. In this study, we aimed to elucidate the comprehensive genetic alteration profile of early-stage breast tumors among Chinese patients. Methods: Surgical tissue samples from 589 Chinese women with stage I-III breast cancer with various histology and molecular subtype consecutively diagnosed at Guangdong Provincial People’s Hospital were sequenced using a panel targeting 520 cancer-related genes spanning 1.64Mb of the human genome. Clinical and genomic data from our cohort was compared with publicly-available data from 1,046 stage I-III breast cancer patients from the TCGA dataset. Results: Based on the analysis of the genetic alteration profile from our cohort, at least one genetic alteration was observed from 98% of the tumor samples, with TP53 (47%), PIK3CA (45%), ERBB2 (30%), and CDK12 (18%) as the most commonly altered genes. The most common genetic alteration types were copy number amplification (43.6%) and missense mutations (36.8%). As compared with the TCGA dataset, our cohort is mostly composed of women 50 years and younger (59.1% vs. 30.4%, P< 0.001), with significantly fewer patients with lobular carcinoma histology (2.4% vs. 19.0%, P< 0.001), and significantly more patients with pathologic stage I tumors (23.3% vs. 17.3%, P= 0.012). Consistently, genetic alterations detected from our cohort affected genes involved in PI3K (63% vs. 56%, P= 0.009) and cell cycle (23% vs. 35%, P< 0.001) pathways, with statistically different genetic alteration rates as compared with the TCGA dataset. Comparison of genetic alteration profile between the two cohorts revealed that our cohort had more frequent genetic alterations in genes including PIK3CA ( P< 0.001), TP53, particularly in hotspot mutations Q192* ( P< 0.001) and A307V/del ( P= 0.02), and ERBB2 amplification ( P< 0.001). Conclusions: Our study contributes to the understanding of the key pathways and specific genetic alterations harbored by Chinese patients with early-stage breast cancer that could potentially be developed as markers of treatment response to targeted therapeutics.

553 Background: HER2 expression or amplification qualify patients to receive targeted therapeutics against HER2; however, traditional methods of quantifying HER2 amplification using fluorescence in situ hybridization (FISH) do not include a reliable definition for low level amplification. With the promising response rate of patients with low HER2 amplified-metastatic breast cancer to subsequent-line trastuzumab deruxtecan (DS-8201a) therapy, there is a need to improve the existing criteria to accurately identify patients with low HER2. In our study, we investigate whether HER2 amplification quantified by NGS could provide a method to stratify patients into subgroups. Methods: A total of 774 patients diagnosed with breast cancer from Guangdong Provincial People's Hospital (GDPH) who underwent targeted NGS using 520 or 33 cancer-related genes and had their HER2 status evaluated with either FISH or IHC were included in this study. HER2 status were defined as per 2018 ASCO/ACP guidelines. Results: Our results demonstrate that NGS could quantify HER2 amplification with high sensitivity and specificity, with area under the curve of 0.990 [95%CI: 0.982-0.999]. The receiver operating curve indicated an optimal cut-off of 2.62 copy number (CN) for identifying IHC/FISH HER2-negative status with 97.8% specificity. Meanwhile, the cut-off of ≥ 3.62 CN identified patients with IHC/FISH HER2-positive status with 99.8% specificity. Among the 774 patients, 65.8% (n = 509) had HER2 CN of ≤ 2.62 and were classified as HER2 non-amplified, while 25.8% (n = 199) had HER2 CN of ≥ 3.62, classified as HER2-amplified. The remaining 66 patients (8.5%) had HER2 CN between 2.62 and 3.62, and were the patients with heterogeneous IHC/FISH results, classified using NGS as HER2 low-amplified. Patients with low-amplified (49.0% vs. 38.8%, P < 0.001) and amplified (50.3% vs. 38.8%, P < 0.001) HER2 had significantly more number of copy number amplifications in other gene, including CDK12, RARA, and SPOP (P < 0.001, P < 0.001) than patients with HER2 non-amplified, indicating distinct mutation profile. Conclusions: Our results demonstrate that NGS could provide a more accurate stratification of patients based on their HER2 amplification levels. Patients with low levels of HER2 amplification has a distinct mutation profile, suggesting that NGS could serve as a robust tool to identify patients with HER2 amplification, whether high or low, who could benefit treatment with targeted agents designed against heterogeneous HER2 expression.

554 Background: Limited studies have investigated the molecular underpinnings driving breast cancer development in Chinese younger women. Based from our previous data, more Chinese women are diagnosed with early-onset breast cancer than in the West. In our study, we aim to investigate the comprehensive mutational profile of Chinese women 35 years old and younger (≤35y) diagnosed with breast cancer. Methods: Targeted sequencing was performed on surgically-removed tumor tissues and blood samples collected from 589 women diagnosed with stage I-III breast cancer of various molecular subtypes at the Guangdong Provincial People’s Hospital (GPDH) using a gene panel interrogating 520 cancer-related genes. We compared the data of 53 women aged ≤35y from our cohort to the data from 33 breast cancer patients aged ≤35y included in The Cancer Genome Atlas (TCGA) dataset. Results: Among the women aged ≤35y with early-stage breast cancer from both cohorts, our cohort had more number of hormone receptor-positive (HR+) patients (GPDH, 72% vs. TCGA, 61%, P< 0.001). Analysis revealed an overall mutation detection rate of 98% in our cohort, with mutations affecting genes involved in the PI3K pathway (47%) and cell cycle pathway (23%). TP53 and PIK3CA were the most commonly mutated genes, with mutation rates of 51% and 25% from our cohort. No statistical difference in mutation profile was found between GPDH and TCGA cohorts. Moreover, germline mutations considered as pathogenic and likely pathogenic (P/LP) in breast cancer susceptibility genes including BRCA1 (n = 4), BRCA2 (n = 2), PALB2 (n = 1), PMS2 (n = 1), PTEN (n = 1), and ATM (n = 1) were detected from 18.9% (10/53) of the patients from our cohort. Women aged ≤35y had significantly more germline BRCA1 mutations than patients > 35y from our cohort (7.5%, 4/53 vs. 2.1%, 11/536 P= 0.049). Conclusions: Our study has identified the involvement of PI3K and cell cycle as the two key pathways in the early development of breast tumors in younger women. In addition, our results also support the role of P/LP germline mutations in breast oncogenesis in Chinese patients with early-onset breast cancer, indicating the need to include a more comprehensive germline mutation screening in our population.

543 Background: HER2 targeted therapy has revolutionized the survival outcomes of early and advanced HER2+ breast cancer (BC). However, among HER2+ patients, the therapeutic response to HER2 inhibitors vary. To understand the molecular mechanism of the variability in therapeutic efficacies, the mutational landscape of HER2+ tumors need to be elucidated. Methods: 107 HER2+ Chinese stage I-III BC patients were included in the study, including 64 HR+ and 43 HR- patients. A majority of the patients were diagnosed with infiltrating ductal carcinoma (99/107). Capture-based targeted sequencing was performed using a panel consisting of 520 cancer-related genes spanning 1.64 MB of the human genome. Results: 1,119 alterations were detected, including 478 single nucleotide variants (SNVs), 14 insertions or deletions, 29 fusions, 593 copy number amplifications (CNA), 2 large genomic rearrangements and 3 CN deletions in 267 genes. Alterations in 99 genes were shared between HR+/HER2+ and HR-/HER2+ tumors; while 123 and 45 genes were only detected in either HR+/HER2+ or HR-/HER2+ tumors, respectively. CNA, splice site and frameshift mutations were significantly more in HR+/HER2+ patients ( p= 0.017). Specifically, CNA in SPOP, CCND1, FGF19, FGF3, FGF4, RNF43, RAD51C, ADGRA4 and MDM4 and various alterations in GATA3 were significantly more among HR+/HER2+ tumors ( p< 0.05). In addition to HER2 amplifications, concurrent fusions in ERBB2 (67%, 4/6), SNVs in ERBB3 (100%, 3/3) and ERBB4 (100%, 1/1) were more likely to be detected in HR+/HER2+ tumors, while concurrent EGFR amplifications were exclusively detected in HR-/HER2+ tumors. The trend of concurrent mutations was consistent with mutation types detected in HER2- tumors based on HR status, wherein EGFR amplifications were more frequent in HR-/HER2- tumors, while SNVs in EGFR, ERBB2, ERBB3 and ERBB4 were more predominant in HR+/HER2- tumors. Based on KEGG pathway analysis, HR+/HER2+ tumors had more frequent alterations in TGFb ( p= 0.007), WNT ( p= 0.002) and homologous recombination ( p= 0.004) pathways than HR-/HER2+ tumors. Furthermore, our data revealed that HR+/HER2+ and HR-/HER2+ patients had comparable TMB ( p= 0.24), with a median TMB of 4.0 mutations/Mb for both. Conclusions: Our study revealed genetic heterogeneity between HR+ and HR- HER2+ tumors. The distinct genetic alterations are potentially relevant in the development of optimal treatment strategies for such patients.

579 Background: The standard management of early stage human epidermal growth factor receptor 2 (HER2) positive (+) breast cancer (BC) involves neoadjuvant therapy with combination of chemotherapy and HER2-targeted therapy followed by surgery. However, diverse pathologic responses were observed. We interrogated whether baseline genomic heterogeneity contributes to the varied therapeutic responses. Methods: Capture-based targeted sequencing using a panel consisting of 520 cancer-related genes, spanning 1.6MB of human genome, was performed on tissue biopsy samples, obtained prior to neoadjuvant therapy, of 33 HER2+ women with stage I-III BC. The median age of the cohort was 53. The correlation between genomic alterations and pathologic response were analyzed by multivariate analysis. Results: A majority of them was diagnosed with stage II (67%, 22/33), while 30% (10/33) had stage III and 3% (1/33) had stage I disease. 58% (19/33) were HR+ and 42% (14/33) were HR-. Mutation profiling of baseline samples revealed 349 mutations spanning 145 genes, with TP53, CDK12 and PIK3CA being the top 3 most frequently mutated genes. Neoadjuvant regimen was comprised of trastuzumab and HER2 inhibitor (i.e. pertuzumab or lapatinib). 15 patients used single HER2 inhibitor;18 used dual HER2 inhibitors. Endocrine therapy was also administered to HR+ patients (19/33) in combination with trastuzumab and HER2 inhibitor. Complete pathologic response (pCR) was observed in 45.5% (15/33) of patients. Interestingly, ROS1 copy number amplifications (CANs) were only identified in patients achieved pCR (p = 0.033). In contrast, missense mutations in PIK3CA and CNAs in CCND1, FGF19, FGF3, FGF4, SPOP, HNF1B and BRIP1 showed a trend of being less likely to mutate in pCR patients (p values between 0.05-0.1). Previous reports have suggested that pCR rates in HER2+ patients are associated with HR status. However, our data revealed comparable pathologic response of patients based on either HR status or neoadjuvant regimen. Conclusions: Our data revealed a distinct mutational profile between patients achieved pCR vs patients did not. Further studies with a larger cohort are required to confirm these findings.

e23020 Background: CTCs has been confirmed to associate with a poor outcome of breast cancer at primary diagnosis. To date, CTC has not been studied with sufficient details in the follow-up of non-MBC pts, especially in Chinese population. We investigated the associations of CTC with clinicopathological features and metabolic factors to gain insight into potential interactions between physiology and disease burden. Methods: We recruited 264 cases of postoperative pts with non-MBC in Guangdong General Hospital from Jan 2009 to Dec 2015. For each enrolled pts 7.5mL blood samples were drawn for CTC enumeration using the CellSearch system. CTC results were correlated with clinicopathological features, obesity related indicators and HBsAg. A multivariate logistic regression analysis was performed to further determine the independent predictors of CTC. The study defined CTC positive as CTC ≥1. Results: In 10.6 % of all pts (n = 28) a median of 1 (range, 1–108) CTC was detected. A pT1 tumor was present in 47% of pts, 11.4 % had G1 grading and 64.4 % were node-negative. The positive rate of CTCs in pts with IDC and ILC was lower than that in pts with other pathological types (9% and 11.1% vs 28.6% P=0.02). A similar trend that approached significance was noted for age (p=0.062), histological grade( p=0.087), invasion of the nipple areola (p=0.06). CTCs was positively correlated with blood glucose (p=0.015), and negatively correlated with high density lipoprotein (p=0.049). In addition, glycated hemoglobin was positively associated with CTC in correlations that neared significance (p=0.059). The result of multivariate logistic regression analysis is shown in the Table. No associations were observed in triglycerides, free fatty acid, total cholesterol, low-density lipoprotein and HBsAg. Conclusions: This study suggested pathological type and blood glucose were independent predictors of CTC, supporting the notion that high fasting glucose level in BC pts may have severe disease burden, leading to a higher frequency of CTC. [Table: see text]

Our retrospective study in breast cancer patients evaluated whether integrating subtype and pathologic complete response (pcr) information into axillary lymph node restaging after neoadjuvant chemotherapy (nac) adds significance to its prognostic values. Patients included in the analysis had stage ii or iii disease, with post-nac axillary lymph node dissection (alnd), without sentinel lymph node biopsy before completion of nac, with definitive subtyping data and subtype-oriented adjuvant treatments. The ypN grading system was used to restage axillary lymph node status, and ypN0 was adjusted by pcr in both breast and axilla into ypN0(pcr) and ypN0(non-pcr). Univariate and multivariate survival analyses were performed. Among the 301 patients analyzed, 145 had tumours that were hormone receptor&ndash, positive (hr+) and negative for the human epidermal growth factor receptor (her2&ndash, ), 101 had tumours that were positive for her2 (her2+), and 55 had tumours that were triple-negative. The rate of pcr in both breast and axilla was 11.7%, 43.6%, and 25.5% respectively for the 3 subtypes. Compared with the non-pcr patients, the pcr patients had better disease-free survival (dfs) and overall survival (os): p = 0.002 for dfs and p = 0.011 for os. In non-pcr patients, dfs and os were similar in the ypN0(non-pcr) and ypN1 subgroups, and in the ypN2 and ypN3 subgroups. We therefore grouped the ypN grading results into ypN0(pcr) (n = 75), ypN0&ndash, 1(non-pcr) (n = 175), and ypN2&ndash, 3 (n = 51). In those groups, the 3-year dfs was 98%, 91%, and 56%, and the 3-year os was 100%, 91%, and 82% respectively. The differences in dfs and os between those three subgroups were significant (all p < 0.05 in paired comparisons). Multivariate Cox regression showed that subtype and ypN staging adjusted by pcr were the only two independent factors predicting dfs. Axillary lymph node status after nac, adjusted for pcr in breast and axilla, predicts differential dfs in patients without prior sentinel lymph node biopsy.

1041Background: Nipple-sparing mastectomy (NSM) improved patients’ mental and aesthetics results and showed safe oncologic outcomes. However, there were still controversies about the indications fo...

Trastuzumab-containing neoadjuvant chemotherapy achieves a pathologic complete response (pCR) rate of about 40 % in HER2-positive breast cancers, and pCR predicts better survival. A cohort of 102 consecutive Chinese HER2-positive stage II/III patients with neoadjuvant trastuzumab/taxanes were retrospectively analyzed, to evaluate the role of hormonal receptor (HR) status and Ki67 index, along with other parameters, in pCR and survival prediction. pCR rate of the cohort was 44.1 % (45/102). Fifty-three patients were HR-positive and 49 were HR-negative. Median Ki67 index was 40 %, and 49 patients had a high Ki67 index (>40 %) whereas 53 had a low Ki67 index (≤40 %). HR status and Ki67 index were confirmed as the only two parameters associated with pCR in multivariate analysis (hazard ratio = 2.952; 95 % CI, 1.227–7.105; P = 0.016 for HR status and hazard ratio = 2.583, 95 % CI 1.107–6.026, P = 0.028 for Ki67 index). Patients with coexisting HR-negative and high Ki67 index had higher pCR rate (69.2 %), compared to those with either HR-negative alone or high Ki67 alone (hazard ratio = 3.038; 95 % CI, 1.102–8.372; P = 0.029), and to those with coexisting HR-positive and low Ki67 index as well (hazard ratio = 7.071; 95 % CI, 2.150–23.253; P = 0.001). In a median follow-up duration of 25.9 months, 11 disease-free survival events (DFS) were recorded. pCR predicted better DFS (log rank P = 0.018) and was the only significant factor in Cox regression analysis (hazard ratio = 0.184; 95 % CI, 0.038–0.893; P = 0.036). Our study indicates that HR status and Ki67 index are predictors for pCR but not for DFS in HER2-positive patients with neoadjuvant trastuzumab/taxanes, which deserves further investigations.

The timing of sentinel lymph node biopsy (SLNB) of breast cancer in the neoadjuvant setting is still controversial. We retrospectively analyzed a Chinese patient cohort with neoadjuvant chemotherapy (NAC) to evaluate the accuracy and axilla sparing potentials of different SLNB timings with methylene blue alone for lymphatic mapping.Patients with NAC and axillary lymph node dissection (ALND) and either pre- or post-NAC SLNB were eligible. Clinicopathological characteristics, identification rate (IR), false-negative rate (FNR), accuracy, and positive-predictive value were calculated and compared between the pre- and post-NAC SLNB group using appropriate statistical methods. Axilla sparing potentials of different SLNB timings were evaluated and compared.One hundred and fifteen eligible cases were included, and 58 had pre-NAC SLNB while the other 57 had post-NAC SLNB. Both groups were comparable in clinicopathological characteristics, neoadjuvant treatments and pathologic complete response rate. IR, FNR, and accuracy of SLNB, as pre-NAC versus post-NAC, were 100 versus 98.2 % (P = 0.496), 0 versus 8.0 % (P = 0.181), and 100 versus 96.4 % (P = 0.239), respectively. Post-NAC SLNB had significantly higher positive-predictive value for ALNs than pre-NAC SLNB (70.0 vs. 36.4 %, P = 0.014), suggesting as high as 63.6 % of ALND performed in the pre-NAC group could have been avoided while only 30 % of ALND in the post-NAC group were theoretically unnecessary.Both SLNB timings of breast cancer patients with NAC were feasible and accurate. Although pre-NAC SLNB tends to be better in accuracy, post-NAC SLNB is significantly superior in terms of axilla sparing.

e11532 Background: This study determined the effects of the phosphatase and tensin homolog (PTEN) expression level on autophagic status and on breast cancer's de novo resistance to trastuzumab. Met...

e11529 Background: This study aimed to assess the value of [18F]-FDG PET in predicting pathological complete response (pCR) of breast cancer patients who underwent neoajuvant chemotherapy (NAC). Me...