When evaluating patients with abdominal pain, it is important to consider SAM in the differential diagnosis, along with vasculitis, fibromuscular dysplasia (FMD), atherosclerosis, mycotic aneurysms, and cystic medial degeneration.Segmental arterial mediolysis (SAM) is a rare arteriopathy which is an under‐recognized and commonly missed diagnosis of abdominal pain. We report a case of a 58‐year‐old female who presented with abdominal pain and was misdiagnosed with a urinary tract infection. The diagnosis was made with CTA and managed with embolization. Despite appropriate intervention and close hospital monitoring, further complications were inevitable. We conclude that though literature has shown better prognosis and even complete resolution after medical and/or surgical intervention, close follow up and monitoring is needed to avoid unexpected complications. Urinalysis revealed blood and leukocyte esterase and the patient was discharged on Cephalexin for suspected urinary tract infection. Her abdominal pain did not improve, and she started having nausea, prompting her to visit the emergency department. In the ED, a Computed Tomography (CT) of her abdomen showed findings consistent with recently passed calculus and the patient was again discharged home with a plan for outpatient follow up.She returned after 12 h as her abdominal pain got worse. Her vital signs at presentation were blood pressure 154/80 mm Hg, heart rate 85 beats per min, respiratory rate 17 per min and oxygen saturation of 100% on room air. Physical examination revealed diffuse abdominal tenderness along with right costovertebral angle tenderness. Her initial laboratory test results showed white blood cell count 9.1 bil/L, hemoglobin 13 g/dL, platelets 257 bil/L and INR 4.4. Basic metabolic panel was unremarkable. Liver function panel showed aspartate aminotransferase 658 U/L (compared to 104 U/L 12 h prior), alanine aminotransferase 772 U/L (compared to 82 U/L 12 h prior), bilirubin 0.5 mg/dL, alkaline phosphatase 70 U/L and albumin of 4.6 g/dL. Extensive workup including infectious and rheumatologic testing to understand the etiology of acute elevation in transaminases was grossly unremarkable as in Table An axial CT angiography abdomen/ pelvis with contrast showed new right hepatic lobe infarct measuring 6.5 × 6.5 cm with hypervascular lesion suspicious for pseudoaneurysm of right hepatic artery as in Figure 3SAM was initially described by Slavin et al in 1976 as “Segmental mediolytic arteritis”.The pathophysiology of SAM has been the topic of extensive study and is mainly guided by histologic findings.3, but most studies, reported hypertension in a significant number of patients with the disease.10. A small percentage of patients (4% of patients with unruptured aneurysms) will develop new aneurysms according to Shimohira et al.Most studies concur that SAM most commonly affects men aged 50–60. Most of the studies published agree on the most common presenting symptom being abdominal pain in over two thirds of cases.Diagnosing SAM always poses a clinical challenge.5.Differential diagnosis of this disease is very important, as there are multiple mimics that could make diagnosing SAM particularly challenging.Fibromuscular dysplasia requires special mention, as it shares many similar features with SAM.In the background of medical history such as protein C and S deficiency, deep vein thrombosis, systemic lupus erythematosus, and hypothyroidism in our patient, it is possible that they contribute to vasculopathy. The prevalence of vasculitis in SLE is reported to be between 11% and 36%.10. Most patients will have stable disease, with some having complete resolution of SAM, whereas others will have aneurysm progression or development of new lesions.Treatment of SAM is conservative with blood pressure control, anticoagulation, or antiplatelet therapy,4SAM is a rare non‐inflammatory, non‐atherosclerotic disease of medium sized intra‐abdominal vessels, most commonly affecting men between ages of 50–60. The most common presenting symptom is abdominal pain, and it is usually diagnosed with CTA. Differential diagnosis should include vasculitis, FMD, atherosclerosis, mycotic aneurysms, and Cystic medial degeneration. Most patients presenting with unruptured lesions will need continued surveillance but will not require surgical intervention. Patients with disease progression as well as patients with rupture will need surgical intervention, either endovascular or open surgery, with mortality being around 25%. Clinicians need to have a high suspicion for the diagnosis of this disease, especially if presenting acutely.Ashbina Pokharel: Conceptualization; writing – original draft; writing – review and editing. Ioannis Karageorgiou: Writing – original draft; writing – review and editing. SANGAM SHAH: Writing – review and editing. Madhur Bhattarai: Writing – review and editing. Indira Acharya: Writing – review and editing. Judith Bateman: Writing – review and editing.Authors' have no conflict of interest to declare.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


There is growing evidence for the role of circadian factors in adolescents' sleep and academic adjustment, with greater evening preference being linked to poorer academic functioning. However, studies have yet to evaluate this association prospectively in adolescence, nor have studies examined daytime sleepiness as a putative mechanism linking evening preference to poor academic functioning. The current study used a multi‐informant design to test the prospective association of evening circadian preference, daytime sleepiness, and academic functioning  across 2 years in adolescence. As evening circadian preference, sleepiness, and academic problems are elevated in adolescents with ADHD, we used a sample enriched for adolescents with ADHD and explored whether ADHD moderated effects.Mage = 13.17 years; 44.7% female; 81.8% White; 52% with ADHD). In the fall of eighth grade, adolescents reported on their circadian preference, and in the fall of ninth grade, adolescents and parents completed ratings of daytime sleepiness. In the middle of 10th grade, parents and teachers reported on adolescents' academic impairment and at the end of 10th grade, adolescents' grade point average (GPA) was obtained from school records.Participants were 302 adolescents  and baseline academic impairment, greater self‐reported evening preference in 8th grade predicted increased parent ratings of academic impairment in 10th grade indirectly via adolescent and parent ratings of daytime sleepiness in 9th grade. Furthermore, evening preference in 8th grade predicted greater teacher ratings of academic impairment and lower average GPA in 10th grade via parent ratings of daytime sleepiness in 9th grade, controlling for covariates and baseline GPA. ADHD status did not moderate indirect effects.Findings underscore daytime sleepiness as a possible intervening mechanism linking evening preference to poor academic functioning across adolescence. Intervention studies are needed to evaluate whether targeting circadian preference and sleepiness improves academic functioning in adolescents. There has been growing interest in the role of circadian factors in the development and maintenance of sleep and academic functioning in adolescence  between the ages of 12 and 14 years . Parents identified adolescents' race/ethnicity as European American/White (81.8%), multiracial (7.9%), Black/African American (5.3%), Asian (4.5%), and American Indian/Alaskan (0.3%). Slightly more than half of participants (53%) had a reported family income of $100,000 or higher, 31.2% between $50,000 and $100,000, and 14.5% less than $50,000. Further description of the sample and comparisons can be found elsewhere  and meeting criteria for ADHD or comparison group. Adolescents eligible for the ADHD group met fifth edition of the Diagnostic and Statistical Manual for Mental Disorders (DSM‐5) criteria for ADHD combined presentation or inattentive presentation based on the parent version of the Children's Interview for Psychiatric Syndromes  at Cincinnati Children's Hospital Medical Center and Virginia Commonwealth University. This study uses data from three time points: the fall of 8th grade (T1), the fall of 9th grade (T2), and the fall/winter of 10th grade (T3). School staff at middle schools around Cincinnati, Ohio and Richmond, Virginia were provided with recruitment materials to distribute to families . Parents contacted the research staff in response to recruitment materials and, after meeting general phone screening criteria , were invited for the initial visit. At this visit, the adolescent and parent received a comprehensive assessment, with inclusion criteria including: full scale IQ >80 on Adolescents were evaluated for ADHD at the initial visit in accordance with the DSM‐5 criteria. The parent version of the P‐ChIPS . Parents also provided their perception of adolescents' daytime sleepiness (‘How often does your child get sleepy or drowsy while doing homework?’). The PDSS has well‐documented psychometric properties and convergent validity with sleep problems . Manifest variables were used given the sample size, and all models were just identified (df = 0). Consistent with recommendations on handling nonignorable missing data, we used full‐information maximum likelihood with adolescent age and family income as auxiliary variables as an approach to reduce the impact of the nonignorable missing data biased parameter estimates  = 196.276, p < .001), with participants completing all time points being older, having a higher parent‐reported family income, and more likely to be in the non‐ADHD group (ps < .05). Thus, adolescent age and family income were used as auxiliary variables in missing data estimation moving forward. Primary study variables were normally distributed .There was a small‐to‐moderate amount of participant attrition across the three time points . Furthermore, 3.6% of participants did not have GPA data at baseline, whereas 22.8% did not have GPA at the T3 visit. Little's MCAR test in SPSS indicated that data were missing in a pattern consistent with not missing at random (χps < .05). These variables were included as covariates in primary analyses, with age and family income as an auxiliary variable in missing data estimations.Table p = .014), controlling for covariates and T1 parent‐reported academic impairment. ADHD status was associated with higher T3 ratings of academic impairment . Additionally, T1 evening preference was uniquely associated with greater T2 self  and parent  ratings of daytime sleepiness. Adolescent girls had greater T2 self‐reported daytime sleepiness , and T1 medication status , shorter sleep duration , and parent‐reported academic impairment  were each significantly correlated with greater T2 parent‐reported daytime sleepiness. Finally, T2 self‐  and parent‐reported daytime sleepiness  were uniquely associated with greater parent‐reported T3 academic impairment.The first set of analyses evaluated relations between T1 evening preference, T2 self or parent ratings of daytime sleepiness, and T3 parent‐reported academic impairment. Regarding effects of specific paths, T1 evening preference was uniquely associated with increased T3 parent‐reported academic impairment  and parent  ratings of daytime sleepiness. Similarly, T1 medication status  and shorter sleep duration  were associated with greater T2 parent‐reported daytime sleepiness. T2 parent‐reported daytime sleepiness , but not self‐reported sleepiness (p > .05), were uniquely associated with increased T3 teacher‐reported academic impairment.Second, we tested T1 evening preference, T2 self or parent ratings of daytime sleepiness, and T3 teacher‐reported academic impairment. Consistent with bivariate correlations, T1 evening preference was unrelated to T3 teacher‐reported academic impairments. Similar to the previous model with parent ratings of academic impairment, T1 evening preference was uniquely associated with both T2 self .Finally, as presented in Table p < .001) and parent  ratings of daytime sleepiness. Furthermore, T2 parent‐reported daytime sleepiness , but not self‐reported ratings of sleepiness (p > .05), was uniquely associated with lower T3 GPA, controlling for baseline covariates.Above and beyond covariates and baseline GPA (collected end of eighth grade), there was no direct effect of T1 evening preference on T3 GPA. Similar to the above models, T1 evening preference was uniquely associated with greater T2 self . The indirect effect of T1 evening preference to T3 GPA via T2 self‐reported daytime sleepiness was not significant (p > .05).We then tested the indirect effect of T1 evening preference → T2 self‐ or parent‐reported daytime sleepiness → T3 GPA. As presented in Table p = .017), but not T3 english/language arts, history, or math grades (ps > .05). Furthermore, controlling for baseline covariates and grades, T2 parent‐reported daytime sleepiness was associated with lower T3 History  and Science  grades, but not english or math grades. T2 self‐reported daytime sleepiness was unrelated to T3 grades (ps > .05). Finally, as presented in Table As supplemental analyses, we examined the direct and indirect effect on specific academic subjects. First, controlling for covariates and baseline grades, findings showed a direct effect of T1 evening preference on lower T3 science grades .Across all models, ADHD group status did not moderate the indirect effect of circadian preference on academic impairment, overall GPA, or specific academic subjects , multi‐informant  design to test the prospective association of evening circadian preference, daytime sleepiness, and academic outcomes across 2 years in a sample of adolescents with and without ADHD. First, above and beyond demographic factors, ADHD group status, medication status, sleep duration, and baseline ratings of academic outcomes, factors shown in previous research to be associated with academic outcomes  and science (per correlation and direct effect results) grades were small in magnitude. Furthermore, findings from the direct and indirect effect of evening preference on specific grades were not consistent and findings should be interpreted cautiously. Still, a few studies have shown unique effects of sleep disturbances and sleepiness on verbal abilities and complex cognitive functions compared to nonverbal/automatic processes (Buckhalt, El‐Sheikh, & Keller, Finally, the magnitude of indirect effects did not differ for adolescents with and without ADHD. Although surprising at first given documented elevations in evening preference, daytime sleepiness, and academic impairments in adolescents with ADHD (Langberg et al., specific underlying neurocognitive and academic processes underlying this developmental sequence. Regarding the synchrony of circadian preference and peak performance (Goldstein et al., Collectively, our findings highlight the role of daytime sleepiness as an intervening mechanism linking circadian evening preference with academic outcomes. With few exceptions, these findings indicate that evening preference and academic outcomes may not be related directly but through their relationship with daytime sleepiness (Mathieu & Taylor, Despite a number of strengths, including a multi‐informant examination of the longitudinal association of circadian preference, daytime sleepiness, and academic impairment over 2 years in adolescents with and without ADHD, a few limitations are important to note. First, our prospective observational study precludes drawing causal influences of evening preference on sleepiness and academic performance. Thus, we encourage future research to consider implementing experimental designs to better understand mechanisms linking evening preference to increased sleepiness and, in turn, poor academic outcomes. Specifically, similar to studies examining effects on cognitive abilities (Goldstein et al., Findings from the current study are the first to identify daytime sleepiness as a mechanism linking evening circadian preference and academic outcomes across a 2‐year period in adolescence. Specifically, self‐reported evening circadian preference was associated with increased parent ratings of academic impairment through self‐ and parent‐reported daytime sleepiness, whereas parent ratings of daytime sleepiness explained the link between evening preference and increased teacher‐reported impairment and lower GPA, in addition to lower grades in english/language arts and history subjects specifically. These findings highlight the continued need for longitudinal mediation studies to better understand how circadian factors impact academic adjustment in adolescents.


TTN and PLEC, and dozens of metastasis‐related genes, such as FOXA1, NCOA1, CD34, and BRCA2. Ras signalling and arachidonic acid metabolism were uniquely enriched in metastatic cancer. Gene programmes 10 and 11 showed the signatures indicating the occurrence of metastasis better. A module (135 genes) was specifically associated with metastasis. Of them, 67.41% reoccurred in program 10, with 26 genes further retained as the signature genes related to PCa metastasis, including AGR3, RAPH1, SOX14, DPEP1, and UBL4A. Our study provides new molecular perspectives on PCa metastasis. The signature genes and pathways could be served as potential therapeutic targets for metastasis or cancer progression.Prostate cancer (PCa) has a certain degree of heritability, and metastasis occurs as cancer progresses. However, its underlying mechanism remains largely unknown. We sequenced four cases of cancer without metastasis, four metastatic cancer, and four benign hyperplasia tissues as controls. A total of 1839 damaging mutations were identified. Pathway analysis, gene clustering, and weighted gene co‐expression network analysis were employed to find characteristics associated with metastasis. Chr19 had the most mutation density and 1p36 had the highest mutation frequency across the genome. These mutations occurred in 1630 genes, including the most frequently mutated genes  In addition to sharing some key driver mutations with the primary tumour from which they arise, metastatic tumours often develop additional mutations as they evolve during metastasis and treatment.In recent years, next‐generation sequencing‐based analysis has greatly improved our understanding of the genomic definition and molecular characteristics of cancer. Large cohorts like The Cancer Genome Atlas (TCGA), Chinese Prostate Cancer Genome and Epigenome Atlas,In this study, PCa and PCa metastasis samples were deployed for whole exome sequencing (WES) and transcriptome sequencing. Both genomic and transcriptomic landscapes for PCa metastasis were characterized. More importantly, signature genes related to PCa metastasis were further identified.22.1Twelve tissues were obtained from Yuhuangding Hospital, including four prostatic hyperplasia samples, four PCa samples, and four PCas with multiple metastases. All tissues were collected at the time of puncturing. Pathological diagnoses were conducted by two independent and expert pathologists. All tumour tissues were confirmed as prostate adenocarcinoma. The specimens were stored at −80°C until sequencing. None of the cancer patients received any therapy before their diagnosis. The Yuhuangding Hospital Ethics Committee approved the study protocol. All experiments complied with the relevant regulations, and all patients provided written informed consent.2.2Total genomic DNA was extracted and mechanically fragmented  on the Covaris S220 Focused‐ultrasonicator (Covaris). Quality control was performed using the Agilent Bioanalyzer 2100 (Agilent Technologies) to ensure an average fragment size of 150–200 bp. End repair, A‐tailing, adaptor ligation, purification, and enrichment of DNA fragments were then performed. A 225‐ to 275‐bp band was selected, and exome capture was performed using the SureSelectXT Reagent Kit (Agilent Technologies). The DNA library was quantified using the Qubit Fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). Samples were subjected to paired‐end sequencing using the Illumina NextSeq 500 platform with a 150‐bp read length. Fastp (v0.19.5)2.3The variants were filtered with the following criteria: (1) variants classified as stop‐gain or stop‐loss, (2) variants classified as frameshift and nonsynonymous in cancer samples, (3) variants with minor allele frequency less than 1% in the human population , (4) variants predicted to be deleterious in at least three of five in silicon analysis , of which the criterion for CADD was greater than 20.2.4Total RNA was extracted using the TRIzol reagent according to the manufacturer's protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Then the libraries were constructed using TruSeq Stranded mRNA L.T. Sample Prep Kit (Illumina) according to the manufacturer's instructions. The libraries were sequenced on an Illumina HiSeq X Ten platform by O.E. Biotech Co., Ltd., and 150 bp paired‐end reads were generated.Raw reads were first processed using Trimmomatic (v0.36),2.5p value <0.05 and |log2FC| > 1.The factoextra package in R (v4.1.2) was used to complete PCA. The DESeq2 (v1.34.0)2.6https://hiplot‐academic.com/basic/gene‐trend) was used to classify the different expression patterns.Genes were divided into 11 clusters with K‐means in R (v4.1.2). The average expression of each gene  in each group was calculated. The Gene cluster trend module in Hiplot Webgestalt (2.8x + 1) and used as input for the Weighted Gene Co‐expression Network Analysis (WGCNA).We first removed genes whose expression value (FPKM) is equal to 0 in all samples. The filtered data was log‐transformed with log2(3WES and RNA‐Seq were performed to profile gene mutation and expression from four prostatic hyperplasia patients (as control group P), four PCa patients (PC group), and four PCas with multiple metastases (M_PC group). None of the cancer patients received any therapy before their diagnosis. We generated over 1.22 billion raw reads and an average of 100 million per sample from WES. The coverage of the sequencing depth ranged from 82.42 to 113.20X, with an average depth of 96.24X. We acquired 556.92 million raw reads from RNA sequencing in total and an average of 46.41 million for each sample. The WES and RNA sequencing statistics are summarized in Table 3.1in silico prediction  kinases involved in cellular stress responses and migration in cancer. High levels of PLEC are associated with PCa and metastasis.In alignment with the previous findings,FOXA1, NCOA1, BRCA1, EPHA6, SPOP, etc.  than in TCGA PCa (4%).FOXA1 were identified in a single M_PC patient. One mutation in CD34 (rs28362497:C>A) was identified in the M_PC group. CD34 is observed as one of the hallmarks indicating tumour progression and metastasis.CD34 confers tumour progression and aggressiveness on PCa accompanied by higher prostate‐specific antigen level, Gleason score, and the possibility of tumour recurrence.Some PCa genes were also identified as having damaging mutations, such as tc. Data . As a pi3.2p < 0.05 and |log2FC| > 1) were then identified using the DESeq2 package, with a total of 4005, 2925, and 102 yielded from each comparison NOS3 played a relatively crucial role in these selected genes. NOS3  was highly expressed in metastatic PCa and was targeted by microRNA to suppress bone metastasis.CST2 and PGM2L1 functioned in the cell migration and invasion of PCa.AGR3 in cancer development remains unclear. However, AGR3 could bind to metastasis‐related proteins C4.4a and DAG‐1, and co‐expression analysis depicted some AGR3 partners linking AGR3 with cell adhesion, tight junction, motility, metastasis, and regulation of cell cycle.RAPH1, SOX14, DPEP1, and UBL4A have a significant role in the invasion or metastasis of breast cancer, cervical cancer, colon cancer, and pancreatic ductal adenocarcinoma, respectively.LHX6, a tumour suppressor gene, as a factor in colorectal adenocarcinoma.LHX6 was observed down‐regulated in PCa cell lines2FC = 1.79, padj = 0.04).We further explored the 26 genes under the specific phenotypes and diseases (keywords: prostate cancer and metastasis) in the Phenolyzer .Previous research has reported some essential molecules in PCa.TP53 and KMT2C having a higher mutation rate  were differentially expressed (padj < 0.05) in PC or M_PC. ETV4, WNT10A, and AXL were down‐regulated in PC, while FOXA1 and KMT2C were up‐regulated in PC. In M_PC, FOXA1 was over‐expressed, while WNT10A and WNT4 were down‐regulated. FOXA1, with damaging mutations in M_PC2, was the sole one over‐expressed in PC and M_PC simultaneously. In contrast to FOXA1, WNT10A showed the most down‐regulation in both PC (log2FC = −4.29) and M_PC (log2FC = −3.32). On the other hand, 13 genes showed expression differences but without damaging mutations  were down‐regulated in PC and three  were down‐expressed in both PC and M_PC. ETS family showed a downward trend no matter in PC or M_PC.Only 21 genes harboured damaging mutations, with 4With the development of PCa, metastasis will eventually happen to patients, and metastases are the final result of a multi‐stage process. This process depends on the specific phenotypic characteristics of tumour cells and the interaction with the host microenvironment and immune system. Genetic alterations in primary and metastatic tumours may contribute to these phenotypes and host interactions.WNT4 located in 1p36 and the induction of WNT4/TCF7L1 resulted in increased malignancy in prostate cancer that was linked to dysregulation of androgen receptor signalling and activation of the IL‐8/CXCR2 pathway.CCND1 (11q13). CCND1 was over‐expressed in more aggressive prostate cancer phenotypes, and its expression was regulated by oestrogens via ERβ and might contribute to the progression and pathogenesis of prostate cancer.KMT2D and BRCA1 mutation frequency were higher in metastatic samples.KMT2D and BRCA1, their expressions were found significantly higher in M_PC. Similar situations happened to other vital genes such as EZH2, CDKN2A, and ETV5. From this perspective, we could see that the modifications of genes were not wholly consistent with previous studies. Although genes might have multiple forms of mutations in different types of samples, their expression differences would intuitively be reflected at the RNA level, which also showed the necessity and advantage of multi‐omics combination on the other hand.With the assistance of WES, we discovered that some chromosomal regions were highly mutated. Chr1 had the highest frequency within the filtered 1839 variants. ALOX15 (LOX15 or 15‐LO‐1) was found highly expressed in M_PC and PC groups. One of the methylated CpG dinucleotides in the ALOX15 promoter was correlated with microdissected high‐grade prostatic intraepithelial neoplasia, PCa and metastatic human prostate tissues indicating ALOX15 was a valuable marker for disease initiation and progression.GPX3 was clearly down‐regulated in PCa which was consistent with the previous result.GPX3 was negatively associated with PCa progression and its overexpression inhibited the PCa invasiveness and the expression of c‐met.Our pathway analysis for the DEGs revealed several common pathways between PC and M_PC, which have already been identified as participants in various cancers, such as the PI3K‐Akt signalling and MAPK signalling pathway. More importantly, we found two pathways, the A.A. metabolism pathway and Ras signalling pathway, were specifically enriched in the M_PC group. The A.A. metabolism pathway has been proven essential in developing and progressing malignant PCa. As a member of the A.A. pathway, CST2 functioned in the migration and invasion of PCa cells with a crucial role in cell adhesion molecules, extracellular matrix‐receptor interaction and focal adhesion.PGM2L1 was involved in multiple processes in PCa, including glucose consumption, lactic production, cell migration and invasion.DPEP1 was prominently up‐regulated in colorectal cancer during the transition from low‐grade to high‐grade intraepithelial neoplasia and functioned as a positive regulator for metastasis by regulating E‐cadherin expression.DPEP1 was negatively associated with the histological grade and invasion of tumour cells.LHX6 inhibits tumorigenesis and progression in multiple cancers by regulating various pathways or genes which were involved in proliferation, apoptosis, migration, and cell cycle.LAGE3 promoted migration and invasion of hepatocellular carcinoma by facilitating the JNK and ERK signalling pathways.UBL4A inhibits metastasis of pancreatic ductal adenocarcinoma.To clarify gene expression change in different groups, we performed clustering analysis, and part programmes presented high expression in M_PC. There was an upward tendency in program 11, and pathway analysis suggested that genes in program 11 were related to DNA replication and cell proliferation. Besides, we used WGCNA to combine phenotype and expression and connected with clustering results to narrow programmes. Twenty‐six genes were found by selecting the pattern and module that best matched the feature of metastasis. Among them, several genes had been identified to play a role in the progression and metastasis of PCa, and the remaining were associated with other cancers. 5In summary, our WES and RNA‐Seq analysis provides several novel signature genes and pathways that potentially involve in PCa metastasis. These findings extended our understanding of the molecular mechanisms underlying the metastasis of PCa and would be ultimately beneficial for future prevention, diagnosis, and treatment.Chongjun Xiang: Data curation (supporting); formal analysis (lead); software ; visualization ; writing – original draft . Yue Li: Formal analysis ; writing – original draft . Wenting Wang: Data curation (lead). Huiying Tao: Software . Ning Liang: Visualization . Shuang Wu: Resources . Tianxi Yu: Resources . Xin Cui: Resources . Yaqi Xie: Resources . Hongwei Zuo: Resources . Chunhua Lin: Conceptualization ; funding acquisition ; project administration (lead); supervision . Fuyi Xu: Conceptualization ; funding acquisition ; methodology (lead); project administration (supporting); software ; supervision ; writing – review and editing (lead).This research was funded by Taishan Scholar Program (grant number Tsqn202103198); Natural Science Foundation of Shandong Province (grant number ZR2019MH132); Yantai Key Research and Development Project (grant number 2020MSGY079 and 2019MSGY135); Binzhou Medical University Research Start‐up Fund (grant number 50012305190).The authors confirm that there are no conflicts of interest.The sequencing data have been already submitted to Sequence Read Archive (SRA) of NCBI and were available at PRJNA883010.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Data S1.Click here for additional data file.Data S2.Click here for additional data file.


Prehospital tracheal intubation (TI) is associated with increased mortality in patients with penetrating trauma, and the utility of prehospital advanced airway management is debated. The increased incidence of deadly violence in Sweden warrants a comprehensive evaluation of current airway management for patients with penetrating trauma in the Swedish prehospital environment and on arrival in the emergency department (ED).This was an observational, multicenter study of all patients with penetrating trauma and injury severity scores (ISSs) ≥ 15 included in the Swedish national trauma register (SweTrau) between 2011 and 2019. We investigated the frequency and characteristics of prehospital and ED TI, including 30-day mortality and patient characteristics associated with TI.p < 0.001) than TI in the ED, even when adjusted for ISS . Hemodynamic collapse (≤ 40 mmHg) and low GCS score (≤ 8) were the characteristics most associated with prehospital TI. Traumatic cardiac arrests (TCAs) occurred in 154 (18.9%) patients, of whom 77 (50%) were intubated prehospitally and 56 (36.4%) were intubated in the ED. A subgroup analysis excluding TCA showed that patients with prehospital TI did not have a higher mortality rate than those with ED TI, OR 2.07 , with OR 1.39  when adjusted for ISS.Of 816 included patients, 118 (14.5%) were intubated prehospitally, and 248 (30.4%) were intubated in the ED. Patients who were intubated prehospitally had a higher ISS, 33 , than those intubated in the ED, 25 . Prehospital TI was associated with a higher associated mortality, OR 4.26 (CI 2.57, 7.27, Prehospital TI was associated with a higher mortality rate than those with ED TI, which was specifically related to TCA; intubation did not affect mortality in patients without cardiac arrest. Mortality was high when airway management was needed, regardless of cardiac arrest, thereby emphasizing the challenges posed when anesthesia is needed. Several interventions, including whole blood transfusions, the implementation of second-tier EMS units and measures to shorten scene times, have been initiated in Sweden to counteract these challenges.The online version contains supplementary material available at 10.1186/s13049-023-01151-4. Trauma is a leading cause of mortality and morbidity in the young and healthy., 2 GunshDelay, or failure in securing an airway is a preventable cause of death in trauma patients,–8 and emThe aims of this study were to present the characteristics associated with TI, its associated causes of mortality and to compare prehospital and ED intubations in patients with penetrating trauma in Sweden between 2011 and 2019, during which time the incidence of gun violence increased. We analyThis was a retrospective, descriptive multicenter study of all patients with penetrating trauma and an injury severity score (ISS) ≥ 15 who were registered in SweTrau between its inception on June 13, 2011, and December 31, 2019 . Data are presented as medians with interquartile ranges (IQRs) for continuous variables and numbers and percentages for categorical variables. Continuous data were compared using Wilcoxon rank sum test. Mortality associated with TI and ISS was tested using restricted cubic spline. Dichotomous variables were created for prehospital TI, Glasgow Coma Scale (GCS) score (3–8/9–15), hypo- and hyperventilation (respiratory rate [RR] ≤ 9/ > 29), and severe head and/or thoracic trauma (severity ≥ 3 of AIS code head and/or thorax) and used in a multivariable logistic regression analysis. The results were reported as ORs and 95% confidence intervals (CIs). Variables were selected based on patient characteristics with plausible associations with increased mortality. Statistical significance was set as The study analyzed 4965 patients with penetrating trauma between 2011 and 2019, and 816 patients (16.4%) met the inclusion criteria.n = 75), of prehospital TI were performed without medications, and 36% (n = 43) were performed with medications, compared with TI in the ED, where 84% (n = 208) of intubations were with medications and 16% (n = 40) were without medications (Table p < 0.001) longer when prehospital TI were required  compared with patients without prehospital TI .The median age was 29 years for patients with prehospital TI and 30 years for patients intubated in the ED Table . Stab won = 90) for patients with prehospital TI, 21% (n = 117) for patients without prehospital TI, 42% (n = 105) among patients with TI in the ED, and 22% (n = 107) for patients without TI in the ED (Table p < 0.001), which was reduced when adjusted for ISS, OR 7.84 . Survival as a function of ISS in patients with and without prehospital TI is shown in Fig. p = 0.37)  compared with TI in the ED, even after adjustment for ISS OR 2.88 . The Glasgow Outcome Scale (GOS) score was generally higher for patients who were intubated in the ED than for those with prehospital TI.The 30-day mortality was 76% (n = 64), of prehospital TI were performed by physicians with an associated mortality of 70% (n = 45), and 44.9% (n = 53) were intubated by nurses with a mortality of 85% (n = 45). The median ISS was 32  for patients intubated by physicians and 35  for patients intubated by nurses. The most common characteristics associated with prehospital TI were hemodynamic collapse (≤ 40 mmHg) and low GCS score (≤ 8)  of patients were registered as TCA, of whom 50% (n = 77) were prehospitally intubated and 36.4% (n = 56) were intubated in the ED. Nine patients (5.8%) who suffered TCA survived: 3 with prehospital TI, 5 with intubation in the ED, and 1 without TI.The majority (99%) of patients with a SBP ≤ 40 mmHg and a GCS score ≤ 8 suffered TCA. In total, 18.9% (n = 15) for prehospital TI, 9.2% (n = 45) for patients without prehospital TI, 23% (n = 35) for TI in the ED and 6.5% (n = 21) for patients without TI in the ED. There was no significantly increased mortality associated with prehospital TI with TCA excluded  compared with intubation in the ED, with OR 1.39  when adjusting for ISS. When TCAs were excluded, prehospital TI had a significantly increased mortality compared with patients without prehospital TI , even when adjusting for ISS   compared with ISS (OR 1.07) and GSW (OR 7.08).[p  = 0.068). The rationale for this subgroup analysis was the finding that the majority (64%) of prehospital TI was performed without medication, compared with 16% in the ED, as TI without medication is likely to be performed in a situation of cardiac arrest.In this observational multicenter study, we showed that prehospital TI was associated with a higher 30-day mortality rate than intubations performed in the ED, even after adjustment for ISS. This was specifically related to TCA, and in patients without cardiac arrest, prehospital TI did not affect the mortality rate when compared with intubations in the ED. Previous studies have shown that increased mortality was associated with prehospital advanced airway management 69.2%) when compared with in-hospital airway management (35.9%) in patients with penetrating trauma. Taghavi OR 7.08). A meta-a.2% when 2, and normal respiratory rates may reduce pCO2 with subsequent further reductions in venous return, mean arterial pressure and cardiac output.[A total of 154 18.9%) patients suffered from TCA, 133 (86.4%) of whom required TI either prehospital or in the ED, and 9 (5.8%) patients survived. The frequency of cardiac arrests was substantially higher than previously reported., 17 As r% patientc output..Patient parameters associated with prehospital TI were analyzed using multivariate models, with and without TCA. Hemodynamic collapse (≤ 40 mmHg) and low GCS score (≤ 8) were the parameters most associated with prehospital TI with TCA included, compared with low GCS score (≤ 8) and hypotension ≤ 90 mmHg) with TCA excluded. We have previously shown that a substantial proportion of these patients suffered head injuries, which can also be reflected in the low GCS score. Hypotens0 mmHg wiPrehospital anesthesia in hemodynamically unstable penetrating trauma patients is challenging. Emergency medical services are heterogeneous in organization and available competence, which therefore complicate comparisons.–37 NotabThis study has some limitations to be discussed. First, it is an observational study with inherent biases, and inferences regarding the causation of prehospital TI and mortality could not be made. Second, SweTrau is primarily a trauma registry, and quality indicators of airway management were not registered, which limits the analysis. Third, the coverage of SweTrau increased during the study period, which could confound incidence. However, we did not analyze incidence trends. Fourth, the transcription of ordinal data (blood pressure) limits the multivariate analysis, which was primarily intended as a visualization of associations. Fifth, as registration in SweTrau was limited to patients who arrived at a participating hospital, prehospital deaths are a possible source of selection bias.Prehospital TI was associated with a higher mortality rate than intubations performed in the ED, which was related to TCA; intubation did not affect mortality in patients without cardiac arrest. The mortality rate was substantial when airway management was needed, regardless of TCA, demonstrating the lethality of severe, penetrating trauma and the challenges posed when anesthesia is needed. Several interventions, including whole blood transfusions, implementation of second-tier EMS units and measures to shorten the scene time, have been initiated to counteract challenges with penetrating trauma in Sweden. The effects of these new interventions will be analyzed in future studies.Additional file 1: Figure 6. Flowchart of TI with traumatic cardiac arrests excluded. Abbreviations: ED = emergency department, ISS = injury severity score, TCA = traumatic cardiac arrest, TI = tracheal intubation.Additional file 2: Figure 7. Regression model for mortality associated with prehospital TI with traumatic cardiac arrests excluded. Probabilities calculated using the median age (31 years). Abbreviation: GCS = Glasgow coma scale. Additional file 3: Figure 8. Patient characteristics associated with prehospital TI with traumatic cardiac arrests excluded. Abbreviation: GCS = Glasgow coma scale.


Combined treatment using anti‐programmed death‐ligand 1 antibody (anti‐PD‐L1) and platinum‐etoposide is the current standard first‐line treatment for patients with extensive‐stage (ES) small cell lung cancer (SCLC). However, the best treatment for relapsed ES‐SCLC after the first‐line treatment remains unclear. There are some approved chemotherapeutic agents that can be used against ES‐SCLC, and treatment with irinotecan is well established as both a monotherapy and a combined therapy, in combination with platinum. Therefore, we conduct a phase II study with irinotecan in the second‐ or later‐line setting for patients with ES‐SCLC who have been previously treated with combined treatment.2) will be determined according to the type of UGT1A1 gene polymorphism, and the treatment will be discontinued following disease progression, intolerance, withdrawal of patient consent, and based on the investigator's decision. The primary endpoint of the study is the response rate, and the secondary endpoints are overall survival, progression‐free survival, and safety.Our study will enroll total 30 patients who are diagnosed with ES‐SCLC and have experienced disease progression after the combined treatment. Patients will receive irinotecan on days 1, 8, and 15, which will be repeated every 4 weeks. Doses of irinotecan  on November 4, 2021. Schema of IRICO study; study design, dose of irinotecan, endpoints. Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers and tends to progress and disseminate rapidly, resulting in 70%–85% of patients being diagnosed with extensive‐stage (ES)‐SCLC, defined as the presence of metastatic lesions outside the hemithorax at the time of diagnosis.In cases of relapse after platinum‐etoposide treatment, the standard treatment includes a regimen identical to the initial therapy, nogitecan monotherapy,UGT1A1 gene is genetically polymorphic, and individuals who are heterozygous or homozygous for UGT1A1*6 and UGT1A1*28 have reduced ability to produce SN‐38G and delayed SN‐38 metabolism compared to that of the wild‐type.UGT1A1 polymorphism. Although irinotecan therapy has been approved in many countries as a SCLC treatment, the effect of irinotecan after combined treatment with anti‐PD‐L1 and platinum‐etoposide remains unelucidated.Treatment with irinotecan for patients with ES‐SCLC is well established as both a monotherapy and that in combination with platinum.UGT1A1 status after treatment with anti‐PD‐L1 and platinum‐etoposide.Our study is a phase II trial on irinotecan in the second‐ or later‐line setting after combined treatment with anti‐PD‐L1 and platinum‐etoposide against ES‐SCLC to evaluate the effect of irinotecan after treatment with anti‐PD‐L1 plus platinum‐etoposide. We will evaluate the suitability of irinotecan dosage according to the 2 treatment,2 treatment.2 treatment.2 is often used in clinical practice and refractory relapse was included in this study, assuming an overall response rate of 25% as the threshold response rate, a target response rate of 40% was established. Based on an alpha value of 0.2 and a beta of 0.8, the estimated required number of patients was 30.This is a multicenter, single‐arm, phase II study evaluating the efficacy and safety of investigational therapy (single‐agent irinotecan) in recurrent SCLC after treatment with anti‐PD‐L1 and platinum‐etoposide . All participants will provide written informed consent. The study was registered in the Japan Registry of Clinical Trials (no. jRCT 071210090) on November 4, 2021.Presence of histologically or cytologically confirmed SCLC (combined SCLC is eligible).Patients with relapse after anti‐PD‐L1 and platinum‐etoposide treatment for SCLC (both sensitive and refractory relapse cases are eligible).Eastern Cooperative Oncology group performance status (PS) of 0–2.Age ≥ 20 years.No active malignancy  within the last 5 years prior to consent.Signed written informed consent.3/μL, (c) total bilirubin (T‐Bil) ≤1.5 mg/dL, (d) aspartate aminotransferase (AST) ≤100 U/L, (e) alanine aminotransferase (ALT) ≤100 U/L, (f) serum creatinine (Cr) ≤1.5 mg/dL, (g) SpO2 on room air ≥92%.The latest laboratory results within 14 days before enrollment (the same day of the week 2 weeks before enrollment is acceptable) meet all of the following criteria: (a) neutrophil count ≥1500/μL, (b) platelet count ≥100 × 10Interstitial pneumonia that can be detected using chest radiographs.Chest radiation treatment within 28 days.Superior vena cava syndrome that requires urgent radiation therapy.Clinically serious cardiac disease such as angina pectoris, uncontrolled heart failure, or myocardial infarction within 3 months.Uncontrolled diabetes mellitus.Uncontrolled hypertension.Active systemic infection that requires treatment.Pregnant, or possibly pregnant, postpartum in 28 days, breastfeeding.Fever of ≥38.0°C.Mental disorder or symptoms interfering with daily life and inappropriate for enrollment.Receiving continuous systemic administration  of steroids or immunosuppressive drugs equivalent to 11 mg/day or more of prednisolone.Positive for hepatitis B surface antigen.Positive for human immunodeficiency virus antibody (not necessarily examined).Other inappropriate complications.UGT1A1 gene polymorphism , and treatment will be continued until disease progression, intolerance is detected, or the patient withdraws consent, and based on the investigator's decision.Patients will receive irinotecan on days 1, 8, and 15, which will be repeated every 4 weeks. Doses of irinotecan will be determined according to the type of The primary endpoint of the present study is the response rate, and the secondary endpoints are overall survival (OS), progression‐free survival (PFS), and safety. Efficacy will be assessed according to the Response Evaluation Criteria in Solid Tumors version 1.1. PFS is defined as the time from registration to disease progression or death from any cause, whereas OS is defined as the time from registration to the last day confirmed to be alive or dead from any cause. The Kaplan–Meier method will be used to estimate PFS and OS. Survival analysis will be conducted at the end of the follow‐up period.To our knowledge, this is the first prospective study to evaluate the effect of irinotecan after combined therapy of anti‐PD‐L1 and platinum‐etoposide. Our study will provide a convincing option of irinotecan therapy after anti‐PD‐L1 and platinum‐etoposide therapy in treating SCLC if the primary endpoint is achieved.In our previous study, the sequence of chemotherapy following nivolumab in non‐small cell lung cancer enhanced the antitumor activity of cytotoxic chemotherapy.Consistent with the significant associations between the UGT1A1*6 genotype and irinotecan‐induced toxicities in Asian populations, the Japanese Pharmaceuticals and Medical Devices Agency recommends screening patients for UGT1A1*6 and *28 polymorphisms. The US Food and Drug Administration also recommends a reduction in the irinotecan starting dose in patients who are homozygous for UGT1A1*28.In conclusion, our study will demonstrate and re‐evaluate the efficacy and safety of irinotecan after combined treatment with anti‐PD‐L1 and platinum‐etoposide for patients with ES‐SCLC.All authors had full access to the study protocol and took responsibility for its integrity. Conceptualization, Minoru Fukuda; Investigation, Hiromi Tomono. Hirokazu Taniguchi, Minoru Fukuda, Takaya Ikeda, Seiji Nagashima, Kazumasa Akagi, Sawana Ono, Yasuhiro Umeyama, Midori Shimada, Hiroshi Gyotoku, Shinnosuke Takemoto, Yasushi Hisamatsu, Ryotaro Morinaga, Ryuta Tagawa, Ryosuke Ogata, Yosuke Dotsu, Hiroaki Senju, Hiroshi Soda. Katsumi Nakatomi, Fumiko Hayashi, Nanae Sugasaki, Akitoshi Kinoshita; Methodology, Minoru Fukuda; Project Administration, Hirokazu Taniguchi and Minoru Fukuda; Supervision, Hiroshi Mukae; Visualization, Hiromi Tomono. Writing—original draft preparation, Hiromi Tomono and Hirokazu Taniguchi. Writing—review & editing, Hirokazu Taniguchi, Minoru Fukuda, Takaya Ikeda, Seiji Nagashima, Kazumasa Akagi, Sawana Ono, Yasuhiro Umeyama, Midori Shimada, Hiroshi Gyotoku, Shinnosuke Takemoto, Yasushi Hisamatsu, Ryotaro Morinaga, Ryuta Tagawa, Ryosuke Ogata, Yosuke Dotsu, Hiroaki Senju, Hiroshi Soda, Katsumi Nakatomi, Fumiko Hayashi, Nanae Sugasaki, Akitoshi Kinoshita, and Hiroshi Mukae.All the authors declare no potential conflicts of interest related to this work.


A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia‐mediated neuroinflammation and neuronal apoptosis after SCI.Stimulation of microglia with lipopolysaccharide (LPS) to simulate neuroinflammation models in vitro, the effect of Alpinetin on the release of pro‐inflammatory mediators in LPS‐induced microglia and its mechanism were detected. In addition, a co‐culture system of microglia and neuronal cells was constructed to assess the effect of Alpinetin on activating microglia‐mediated neuronal apoptosis. Finally, rat spinal cord injury models were used to study the effects on inflammation, neuronal apoptosis, axonal regeneration, and motor function recovery in Alpinetin.Alpinetin inhibits microglia‐mediated neuroinflammation and activity of the JAK2/STAT3 pathway. Alpinetin can also reverse activated microglia‐mediated reactive oxygen species (ROS) production and decrease of mitochondrial membrane potential (MMP) in PC12 neuronal cells. In addition, in vivo Alpinetin significantly inhibits the inflammatory response and neuronal apoptosis, improves axonal regeneration, and recovery of motor function.Alpinetin can be used to treat neurodegenerative diseases and is a novel drug candidate for the treatment of microglia‐mediated neuroinflammation. Alpinetin alleviates the inflammatory response and neuronal toxicity caused by activated microglia via targeting the JAK2/STAT3 pathway, and ultimately promotes functional recovery in SCI rats. Alpinetin treatment also alleviated neuroinflammation‐mediated abnormal ROS production and MMP changes in neuronal cells, while achieving a neuroprotective effect. In vivo, we demonstrated that Alpinetin can inhibit the inflammatory response and neuronal apoptosis in the damaged area after SCI, thereby promoting axonal regeneration and ultimately improving motor function recovery. These data suggested that Alpinetin may be a potential neuroprotective agent with antineuroinflammatory effects that could be used in the treatment of neurodegenerative diseases.22.1Alpinetin  was purchased from Chemstrong Scientific Co., Ltd. Cy3‐labeled goat anti‐rat IgG, Alexa Fluor 555‐tagged donkey antirabbit/mouse IgG, Alexa Fluor 488‐tagged goat antirabbit/mouse IgG, horseradish peroxidase‐labeled secondary antibodies, antifluorescence quenching agent (containing DAPI), and CCK‐8 reagent were purchased from Beyotime. Lipopolysaccharide (LPS) was purchased from Merck. Anti‐COX‐2, anti‐Iba‐1, anti‐CD68, anti‐GFAP, and anti‐GAPDH antibodies were purchased from Abcam. Primary antibodies against iNOS, p‐JAK2, p‐STAT3, STAT3, β‐actin, Bcl‐xL, GFAP, NeuN, GAP43, and MAP2 were obtained from Cell Signaling Technology. Alexa Fluor 488 phalloidin was also from CST. The anti‐CD11b antibody was bought from Biolegend. The anti‐JAK2 antibody was provided by Affinity. WP1066 purchased from GlpBio. Transwell chamber was purchased from Corning (0.4 μm pores). Calcein AM/propidium iodide (PI) was bought from Solarbio. The JC‐1 MMP assay kit and the ROS fluorescence test kit were from Elabscience. The polymerase chain reaction (PCR) primers were synthesized by Servicebio while the kit for RNA extraction came from EZBioscience. The reagents for quantitative real‐time PCR (qPCR) were purchased from Vazyme.2.2w/v) sodium pentobarbital (40 mg/kg). The rats were randomly divided into three groups, namely, a SCI (model) group, an Alpinetin group, and a Sham group. In the model group, the spinal cord was exposed with T9 as the center and a crush injury was generated in spinal cord tissue by clamping with a vascular clip for 10 s. In the Alpinetin group, Alpinetin (30 mg/kg) was intraperitoneally injected immediately after SCI. In the Sham group, the spinal cord was exposed, but no injury was made. After surgery, the bladder of the injured rats was emptied twice a day. After surgery, rats in the Alpinetin group were intraperitoneally injected with Alpinetin (30 mg/kg) into the spinal cord for 3 consecutive days.For animal experiments, 54 adult female Sprague Dawley rats (~200 g) were obtained from Hunan Silaikejingda Experimental Animal Co., Ltd. For modeling, rats were anesthetized with 1% (2.3Motor function after SCI was assessed according to the Basso Beattie Bresnahan (BBB) locomotion rating scale, footprint analysis, and electrophysiology. BBB scores varied from 0  to 21 . BBB scores were performed at 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days after SCI to assess the extent of recovery of the hind limbs of the rats after injury. Footprint testing was performed 28 and 56 days after surgery for each group of rats. In addition, motor‐evoked potentials (MEPs) of rats were measured on Day 56 post‐surgery. Briefly, after anesthesia, two stimulation electrodes were placed in the cerebral cortex motor area and the recording electrode was inserted into the anterior tibial muscle of the contralateral hindlimb to record the hindlimb response in the rats. Changes in the amplitude of the MEP were recorded.2.4Heart, liver, spleen, lungs, kidneys, and spinal cord tissue were preserved in 4% PFA, paraffin‐embedded, and cut into 4‐5‐μm‐thick longitudinal and transverse sections. After that, the sections underwent Nissl and hematoxylin and eosin (H&E) staining. Images were acquired using a pathology slide scanner (Olympus).2.52 incubator. Cells were passaged and treated at 80% confluence. The medium was changed every 2 days, and the cell growth was observed under the inverted microscope.Procell provided BV2 and PC12 cell lines. The cells were cultured with high‐glucose DMEM supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were kept alive at 37°C in 5% CO2.65 BV2 cells in the upper chamber of the Transwell inserts and 5 × 105 PC12 cells in the bottom chamber of the inserts), the protective effects of Alpinetin on neurons were assessed using immunofluorescence and Western blot.To assess the impact of Alpinetin on microglia‐induced neuronal death in vitro, BV2 microglia and neuronal PC12 cells were co‐cultured using the Transwell system . Before co‐culture, BV2 cells were pretreated overnight with Alpinetin  and then stimulated or not with 1 μg/ml LPS for 24 h, resulting in varying degrees of microglial activation. To avoid any effects of LPS and Alpinetin on PC12 cells, the BV2 cells were first rinsed with PBS, and then co‐cultured with PC12 cells. After 24 h of co‐culture  for 24 h after seeding in 96‐well plates, and then add 10% CCK‐8 reagent to incubate for 2 h. Finally, a microplate reader (Thermo) was used to detect the optical density at a wavelength of 450 nm.2.8A JC‐1 assay kit was used to detect MMP. JC‐1 acts as a fluorescent probe that can quickly detect changes in the MMP, thereby serving as an early marker of cell apoptosis. PC12 cells in the co‐culture system were incubated with JC‐1 reagent for 30 min in the dark. Using an inverted fluorescence microscope (ZEISS), take images of PC12 cells after removing the dye solution.2.9Dichloro‐dihydro‐fluorescein diacetate  was treated with PC12 cells for 30 min in the dark at 37°C. After rinsing three times with buffer solution, the intracellular ROS concentration was checked using an inverted fluorescence microscope (ZEISS).2.10The PC12 cells in the co‐culture system were treated with 1 μM calcein AM and 2.5 μM PI for 15 min at 37°C. Using an inverted fluorescence microscope (ZEISS), take images of live/dead PC12 cells after removing the dye solution.2.11To evaluate the mRNA expression levels of IL‐1β, TNF‐α, COX‐2, and iNOS, RNA was isolated from BV2 cells using an RNA extraction kit, reversed transcribed, and the resulting cDNA was subjected to qPCR. GAPDH was used for normalization. Table 2.12The RIPA lysis buffer was used to separate proteins from tissues and cells. Then SDS‐PAGE was used to separate the protein sample and then transferred to PVDF membrane and 5% skimmed milk for sealing. Then, incubate the membrane overnight at 4°C with primary antibody against iNOS (1:1000), COX‐2 (1:2000), JAK2 (1:2000), phosphorylated (p)‐JAK2 (1:1000), STAT3 (1:1000), p‐STAT3 (1:1000), Iba‐1 (1:2000), CD68 (1:2000), NeuN (1:1000), Bcl‐xL (1:1000), GAP43 (1:1000), MAP2 (1:1000), GAPDH (1:4000), and β‐actin (1:1000). On the second day, the membrane was incubated with horseradish peroxidase bound secondary antibody at room temperature for 1 hour. After washing, membranes were detected applying a chemiluminescent reagent and visualized using Image Lab software (Bio‐Rad). ImageJ was employed for quantitative analysis.2.135 cells/ml onto round glasses (20‐mm diameter) previously placed in a 6‐well plate, treated, and subjected to immunofluorescence staining. Briefly, the primary antibodies against Iba‐1 (1:300), CD11b (1:100), p‐STST3 (1:300), Bcl‐xL (1:200), and phalloidin (1:500) were incubated overnight at 4°C after the cells had been fixed in 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X‐100, blocked with 5% bovine serum albumin.In vitro, BV2 and PC12 cells were seeded at a density of 3 × 10The next day, the cells were incubated with the respective fluorescent secondary antibodies for 2 h at room temperature. After washing, the coverslips containing the cells were placed on slides, an antifluorescence quenching agent (including DAPI) was added, and the coverslips were sealed. Images were captured by confocal laser scanning microscopy  and analyzed using ImageJ software.For tissue sections in vivo, they were dewaxed in xylene, rehydrated in a graded ethanol series, blocked with 5% bovine serum albumin for 2 h at room temperature, and then incubated with primary antibodies overnight at 4°C. For longitudinal sections, the following antibodies were used: anti‐Iba‐1 (1:300), anti‐CD68 (1:500), anti‐GFAP (1:500), anti‐NeuN (1:200), anti‐Bcl‐xL (1:200), anti‐MAP‐2 (1:200), and anti‐GAP43 (1:300). For transverse sections, the antibodies targeted Iba‐1 (1:300), CD68 , and GFAP . The next day, the sections were incubated with Alexa Fluor 555‐ and Alexa Fluor 488‐conjugated secondary antibodies at 37°C for 1 h. Before sealing the slide with a coverslip, add the fluorescence quenching agent (containing DAPI), and then use a confocal laser scanning microscope (Leica) to take an image.2.14http://swisstargetprediction.ch/), SEA databases (http://sea.bkslab.org/), and HERB databases (http://herb.ac.cn/) were used to predict the target proteins of the Alpinetin monomer (PubChem CID: 154279). Additionally, inflammatory disease‐related proteins were identified via the GeneCards, NCBI, and OMIM databases. Target proteins present in both groups were considered to be potential targets of Alpinetin in inflammation. The overlapping target proteins were also uploaded to the KEGG database for the building of a network of signaling pathways. To analyze drug target interactions associated with Alpinetin, the overlapping targets were also imported into the STRING database and Cytoscape for protein–protein interaction (PPI) network construction. Next, key proteins were screened by topological analysis and MCODE clustering analysis. AutoDock Vina software was used for the docking of Alpinetin with the JAK2 protein (protein number: 4IVA) and for calculating the binding free energy.The SwissTargetPrediction . Statistics are deemed significant when 33.1p < 0.05). To further confirm these findings, we performed immunostaining for Iba‐1, a microglial surface marker that is upregulated during SCI‐induced microglial activation.p < 0.05). We further examined the morphology of LPS‐treated microglia after 24 h of exposure and found that the cells displayed an amoeba‐like morphology (shown by the red arrow)  can reportedly activate BV2 cells and can thus be used to construct a model of neuroinflammation in vitro.) Figure . These rNext, we assessed the level of Alpinetin toxicity toward BV2 cells. Alpinetin's chemical structure is shown in Figure p < 0.05). To further confirm these findings, we performed dual immunostaining for CD11b and Iba‐1  to assess the degree of microglial activation. We found that exposure to LPS upregulated the expression of Iba‐1 and CD11b in microglia, indicating that they had been activated. However, Alpinetin pretreatment markedly suppressed these effects . Subsequently, Western blot analysis showed that pretreatment with Alpinetin (50 and 100 μg/ml) blocked the LPS‐induced increase in the protein expression of iNOS and COX‐2 in a concentration‐dependent manner . Overall, these results suggested that APL exerted significant antineuroinflammatory effects by inhibiting the overactivation of microglia and generation of inflammatory mediators.In order to determine the impact of Alpinetin on inflammatory mediators in LPS‐activated microglia, we first used qPCR to assess the expression levels of genes encoding pro‐inflammatory mediators. As shown in Figure 3.2To further explore the molecular mechanisms by which APL inhibits neuroinflammation in microglia, we obtained 142 predicted protein targets of APL through the SwissTargetPrediction, SEA, and HERB databases. Meanwhile, a total of 100 common targets were identified from among the 3678 genes associated with inflammatory diseases obtained through the GeneCards, NCBI, and OMIM databases Figure . Next, Kp < 0.01). To further evaluate the function of JAK2/STAT3 signaling in the Alpinetin‐mediated reduction of pro‐inflammatory mediator release, we pretreated microglia with Alpinetin (0 or 100 μg/ml) and, before applying LPS, the cells were incubated or not with the JAK2/STAT3 pathway inhibitor WP1066 (5 μM) for 2 h. As shown in Figure p < 0.05). Similarly, the combined application of WP1066 and Alpinetin significantly downregulated iNOS and COX‐2 protein levels compared with WP1066 or Alpinetin treatment alone . We further evaluated the effect of Alpinetin on p‐STAT3 localization in activated microglia via immunofluorescence staining. After LPS treatment, STAT3 phosphorylation was enhanced, and an increase in p‐STAT3 nuclear translocation was observed . Mitochondrial homeostasis is required for the maintenance of cellular physiological functions and the disruption of this balance can lead to cell apoptosis.p < 0.05). A similar trend in Bcl‐xL protein levels was detected using Western blotting . We further measured the MMP in the three groups using a JC‐1 assay kit and found that Alpinetin treatment led to a concentration‐dependent recovery of the MMP (reflected by the relative red/green fluorescence intensity) in co‐cultured PC12 cells . It has been reported that abnormal ROS production induces neuronal apoptosis.p < 0.05). Combined, these data confirmed that Alpinetin exerts significant neuroprotective effects by inhibiting activated microglia‐mediated neuronal apoptosis, mitochondrial dysfunction, and ROS production.To evaluate the effect of Alpinetin on neuroinflammation‐induced neuronal apoptosis in vitro, we used a Transwell co‐culture assay  as shown in Figure 3.4To assess the biosafety of Alpinetin administration after SCI, after 56 days of surgery, we collected the main organs  for H&E staining. No significant differences in the morphology of the organs were observed between the Alpinetin and Sham groups Figure , confirmp < 0.05). These results showed that Alpinetin could significantly promote the recovery of motor function in rats following SCI. Figure p < 0.05). This indicated that anterior and posterior limb coordination was significantly improved after Alpinetin treatment. Next, we used electrophysiological analysis to further evaluate functional recovery after SCI in rats of each group . In contrast, the average signal amplitude recorded in the right and left lower extremities of rats in the Alpinetin group was 1.82 mV and 2.01 mV, respectively, significantly higher than that of rats in the SCI group . . These results showed that Alpinetin administration significantly improved the recovery of motor function in rats after SCI.Next, we evaluated whether Alpinetin can promote motor function recovery in rats after SCI using the BBB locomotion rating scale, footprint analysis, and electrophysiology. All the rats exhibited normal hindlimb motor behavior (scores of 21) before injury. Immediately after spinal cord crush injury, however, the lower limbs of the animals developed complete paralysis (scores of 0) Figure . Within p Figure . On day p < 0.05). These data suggested that Alpinetin markedly enhances spinal cord tissue regeneration and reduces the volume of cavities, leading to improved motor function recovery after SCI.The histomorphology of the rat spinal cord after surgery is depicted in Figure 3.5p < 0.05). Furthermore, compared with the Sham group, the expression levels of inflammatory mediators (iNOS and COX‐2) and those of activated microglia‐related proteins (Iba‐1 and CD68) in rats in the SCI group were significantly upregulated; however, these increases were significantly abrogated after treatment with Alpinetin . Together, these results suggested that Alpinetin exerts a significant antiinflammatory effect by inhibiting the expression of inflammatory mediators as well as by reducing microglial overactivation. These results were consistent with the Alpinetin‐associated antiinflammatory effects observed in vitro , consistent with the Nissl staining and the in vitro neuronal apoptosis results  and immunofluorescence staining  analysis indicated that in comparison to SCI group, the Alpinetin group had a significantly greater protein expression level of the antiapoptosis‐related marker Bcl‐xL. In summary, our findings showed that Alpinetin can effectively suppress neuronal death in the damaged spinal cord.To confirm the neuroprotective effect of Alpinetin in vivo, we examined neuronal retention in spinal cord tissue through Nissl staining. On Day 3 post‐injury, there was a large loss of abdominal motor neurons (indicated by the red arrow) in the SCI group relative to that in the Sham group, an effect that was attenuated by Alpinetin treatment Figure . Moreoves Figure . Next, w3.7p < 0.05), while the number of MAP‐2‐positive cells was significantly increased . This indicated that Alpinetin can reduce neuronal loss and promote axonal regeneration in rats following SCI. To further evaluate the extent of axonal regeneration, nerve filament extension in the injured spinal cord was evaluated by GAP43 protein staining. We observed more neurofilaments in GAP43‐positive cells in the injury area of the Alpinetin treatment group than in that of the SCI group, and the neurofilaments broke through the glial scar to extend toward the center of the site of injury . We also detected MAP‐2 and GAP43 protein expression by Western blot and found that the expression levels of both proteins were obviously increased after Alpinetin therapy , in agreement with the results of immunofluorescence staining. Together, these findings confirmed that Alpinetin treatment helps to mitigate neuronal loss and stimulate axon repair after SCI, which improve the restoration of motor function after SCI.The restoration of motor function following SCI was assumed to be largely dependent on axonal regeneration.4Spinal cord injury is a serious, disabling disease that places a huge burden on patients and their families.Studies have increasingly shown that some bioactive compounds extracted from Chinese herbal medicines can have a significant therapeutic effect on SCI.The JAK/STAT pathway consists of two protein families—JAKSs  and STATs —the members of which can be activated by a variety of inflammatory signals, including LPS.5In summary, we showed that Alpinetin inhibits microglial activation and the associated neuroinflammatory response by inhibiting the JAK2/STAT3 pathway. This alleviates neurotoxic factor‐mediated neuronal apoptosis, which ultimately enhances axon regeneration and promotes motor function recovery after SCI Figure . These fS. X. performed the whole study and wrote the manuscript. Y. Z. assisted with animal experiments and statistical analysis. Z. L. and A. L. helped in the methodology. W. T., X. X. and J. N. participated in animal experiments. N. Z. and G. Z. helped in Methodological analysis. J. L. and Z. L. oversaw the project and provided funding.This work was supported by Jiangxi Provincial Central Committee Guides Local Science and Technology Development Project (No. 20222ZDH04095), Jiangxi Province Traditional Chinese Medicine Science and Technology Program (No. 2020A0068), the “Double Thousand Plan” of Jiangxi Province, Natural Science Foundation of Jiangxi Province (Grant No. 20142BAB215046).The authors confirm that they have no conflict of interest.The authors have read the manuscript and agreed to publish.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Figure CaptionsClick here for additional data file.


Bladder cancer is the 13th most common cancer in China with the predominant histologic type being urothelial carcinoma (UC). Locally advanced and metastatic (la/m) UC accounts for 12% of UC and the five‐year survival rate is only 39.4%, imposing a significant disease and economic burden on the patients. The aim of this scoping review is to synthesize existing evidence of epidemiology, the landscape of treatment options and associated efficacy and safety profiles, as well as treatment‐related biomarkers among Chinese la/mUC patients.A systematic search was conducted on five databases  from January 2011 to March 2022 based on the scoping review criteria in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta‐Analysis Extension for Scoping Reviews.A total of 6211 records were identified, and further review resulted in 41 relevant studies that met all criteria. Additional searches were conducted on epidemiology and treatment‐related biomarkers of bladder cancer to supplement the evidence. Among 41 studies, 24 reported on platinum‐based chemotherapy, eight on non‐platinum‐based chemotherapy, six on immunotherapy, two on targeted therapy, and one on surgery. Efficacy outcomes were summarized by line of therapy. Treatment‐related biomarkers including PD‐L1, HER2, and FGFR3 alterations were identified, and the alteration rate of FGFR3 of Chinese UC patients was lower than that of the western patients.Despite chemotherapy has been the main treatment choice for decades, appealing new therapeutic strategies including ICIs, targeted therapies and ADCs were applied in clinical practice. Further research on epidemiology and treatment‐related biomarkers of la/mUC patients is needed given only a limited number of studies have been identified thus far. High genomic heterogeneity and complexity of molecular features were observed among la/mUC patients; thus, further studies are required to identify critical drivers and promote potential precise therapies. This study was conducted with the rigid and comprehensive procedure of scoping review, in accordance with the guideline developed by Joanna Briggs Institute (JBI).2.1Search terms were developed with the guidance of Population, Concept, and Context framework shown in Supplement File, and preliminary searches were conducted to refine the full strategies (Supplement File). Free text words including “urinary bladder,” “urinary,” “urothelial,” “urothelial cell,” “transitional cell,” “bladder,” “urogenital tract,” “upper tract urothelial,” “ureteral,” “urologic,” “renal pelvis,” “urethra*,” “urothelium,” “tumor*,” “tumour*,” “neoplasm*,” “cancer,” “carcinoma*,” “stage 3,” “stage iii,” “stage 3a,” “stage iiia,” “stage 3b,” “stage iiib,” “stage 4,” “stage iv,” “metasta*,” “advanc*,” “relap*,” “recurrence*,” “refract*,” “late‐stage*,” OR “late stage*,” “inoperable,” “locally advanced,” “locally‐advanced” were used. Searches were limited to paper from peer‐reviewed journals published between January 1, 2011, and March 18, 2022, in both English and Chinese bibliographic databases, including PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), and WANFANG.2.2Studies were included if they: (1) focused on Chinese patients  with locally advanced or metastatic urothelial carcinoma, referring to histological or cytological stage in T3/4, or metastatic to pelvic lymph nodes (N+) or to lymph nodes, organs, or bones (M+); (2) were primary studies; (3) reported information related to epidemiology, existing treatment options and associated efficacy and safety, and genomic alterations and testing method of treatment‐related biomarkers based on large cohort studies; (4) were published in peer‐reviewed English journals, and Chinese journals from the list of Peking University Core Journals of China; and (5) were published between January 1, 2011, and March 18, 2022.Studies were excluded if they: (1) included patients with other disease or early stage urothelial carcinoma; or if no specific result reported for la/mUC patients, more than 20% patients were adenocarcinoma, squamous cell carcinoma, and small cell carcinoma; (2) were case studies, review articles, meta‐analysis, protocols, posters, personal opinions, conference abstracts, or laboratory research (both in vivo and in vitro); and (3) were published in peer‐reviewed journals in language other than English and Chinese.2.3All search results were compiled to the citation management tool (EndNote), and duplications were removed. Two levels of screening were conducted by two independent reviewers (C.Y. and C.C.), and any disagreements were resolved by consensus or by discussion of a third senior reviewer (W.H.). After initial screening, author Q.X. developed a standardized data extraction form, including authors, years, article type, baseline characteristics, outcome, and results of all included studies (Supplement File). Data extractions were conducted by two independent reviewers (T.L. and C.Y.) and conflicts were resolved by a third senior reviewer (J.H.), while no attempt was made to assess the risk of bias for individual studies.33.1Among 6211 references screened, 41 unique studies were included after literature screening  or too old mean or median age , and the mean or median ages in most studies were reported between 50 and 59 year (n = 8) and between 60 and 69 years (n = 22). All patients included were la/mUC patients at stage III/IV. Of 26 studies included population with metastasis, median rates of metastasis in visceral, lymph node, and bone were 53%, 54%, and 17%, respectively.The sample size of included studies ranged from 10 to 575, with proportion of males from 51% to 100%. Among 35 studies reported mean or median age, few studies included patients with a relatively young , and seven eligible studies were included.Treatment options and associated efficacy and safety were investigated in all included studies, while no studies on epidemiology or treatment‐related biomarkers based on large cohort of Chinese la/mUC patients were retained. Noticing this literature gap, additional searches of international epidemiology websites, large China cancer registries, and literature databases were conducted to identify the most recent epidemiology data for BC. Since no articles on treatment‐related biomarkers were retained, further investigation loosed the inclusion criteria from large cohort studies to studies reporting any biomarker testing results. Among 41 included studies, biomarkers including PD‐L1 status and HER2 were mentioned in five studies. There were still no studies on FGFR; however, FGFR3 is demonstrated as the only validated predictive molecular marker in the 2021 update of the European Association of Urology Guidelines (EAU)3.2According to the Global Cancer Statistics, the prevalence of BC in Mainland China was 16.3 per 100,000 population in 2020.3.3Among 41 studies, five types of treatment regimens were identified, including platinum‐based and non‐platinum‐based chemotherapy, immunotherapy, targeted therapy and surgery, categorized based on the CSCO guideline.3.3.1Among 24 studies on platinum‐based regimens, 11 studies investigated patients receiving first line of therapy (LOT), two investigated second‐line therapy, and four investigated mixed line of therapy, while seven studies did not report LOT details .p = 0.005), visceral metastasis (p = 0.032), multiple metastasis (p = 0.024), and number of visceral metastasis ≥3 (p = 0.032) were associated with the poor prognosis of mUC patients.Seven studies did not report information on LOT,3.3.2p = 0.007).A total of eight studies were identified to evaluate the efficacy and safety of non‐platinum‐based chemotherapy, as shown in Table p < 0.05) comparing 3D‐CRT combined with GT chemotherapy to regular radiotherapy combined with GT chemotherapy,Three studies evaluated 3D‐CRT combined with chemotherapy compared to chemotherapy with or without two‐dimensional conventional radiotherapy, and all three studies observed a significant difference in ORR between the observation group and control group.3.3.3Immunotherapies were reported in six studies, two of which were single‐arm trials and four of which were retrospective cohort studies.p value not reported). The other study investigating toripalimab enrolled 151 patients with varying PD‐L1 status,p = 0.002; median PFS, 3.7 vs. 1.8 months, p = 0.001). Median OS was not observed significant difference between PD‐L1+ patients and PD‐L1‐ patients .Two single‐arm studies investigating tislelizumab and toripalimab demonstrated different efficacy profiles between PD‐L1+ and PD‐L1‐ subgroups.Within the three retrospective studies, one study retrieved data from the Taiwan registration system which reported outcomes of UC patients receiving first‐line  and second‐line treatment .In terms of safety, treatment‐related adverse events (TRAE) and immune‐related adverse events (irAE) were reported. For grade ≥3 and incidence rates ≥5% TRAE, anemia, thrombocytopenia, neutropenia, leukopenia, skin rash, and hyponatremia were reported. Only two studies reported irAEs.3.3.4p < 0.05).p > 0.05).Two studies Table  investig3.3.5A retrospective study investigated 23 patients receiving percutaneous cryotherapy who refused systemic chemotherapy or failed previous chemotherapy , next‐generation sequencing , and whole‐genome sequencing (n = 1).In seven studies related to FGFR, five reported FGFR3 alterations,FGFR3 mutations of UC originated in different tumor locations have been investigated, its mutations in UTUC and UC patients were reported in three studies, while no significant difference in proportions was identified.p < 0.001).FGFR3 mutation rates were reported in six studies,4This scoping review aims to understand Chinese la/mUC patients covering the topics of epidemiology, effectiveness and efficacy regarding available treatments, and treatment‐related biomarkers. Through conducting comprehensive literature review and qualitative analysis of reviewed studies, we summarized up‐to‐date incidence, prevalence, and mortality rates of bladder cancer of China. In addition, by investigating the treatment landscape for la/mUC, including platinum‐based chemotherapy, non‐platinum‐based chemotherapy, immunotherapy, targeted therapy, and surgery, this review offered evidence‐based support to help clinicians with their treatment decision‐making. Furthermore, treatment‐related biomarkers specified for Chinese patients, which was a rarely published area, were revealed.Although comprehensive database searches and review of national and international websites were conducted to collect the estimates of epidemiology data, little data were retrieved on la/mUC patients, indicating future studies based on large databases or national cancer registry data are required in China.A variety of treatment options were observed from the search, while platinum‐based chemotherapy was identified to be the major treatment for la/mUC patients in China. The treatment outcomes of first‐line platinum‐based chemotherapy showing the ORR ranged from 41.2% to 77.51% and median OS ranged from 13.5 to 18 months.Most included studies focused on immunotherapy published from 2020 to 2022, implying an increasing investigation in ICIs since the first ICI indicated for la/mUC approved in China. For treatment‐naive and treatment‐experienced patients, ORR ranged from 24% to 54.5% and median OS ranged from 9.8 to 20.53 months.It was found that a substantial proportion of la/mUC patients cannot experience durable responses to ICIs,Published reviews possessed different opinions regarding PD‐L1 status as a response biomarker of ICIs.FGFR alterations have been detected in a wide variety of cancers, while the highest frequency was observed in UC, presenting in 17.9% to 32% among all cancer patients with FGFR alterations.Several ADCs were generated, including RC48‐ADC, enfortumab vedotin, and sacituzumab govitecan. RC48‐ADC, a HER2‐targeting ADC, was approved in China for HER2 overexpression la/mUC patients who failed chemotherapy, in which HER2 overexpression is defined as HER2 IHC2+ or 3+.Admittedly, this study has several limitations. First, quality assessment of included studies was not conducted due to the nature of comprehensive inclusion for literature diversity from scoping review study. Second, comparisons of the efficacy and safety across studies were difficult to be made because of differences in study design, data reporting such as missingness in LOT information. Third, 13 studies included less than 50 patients and 31 studies were conducted more than five years ago, which may not be reflective to current treatment patterns. In addition, since no epidemiological study meeting the inclusion criteria was identified, relevant websites have been searched, which reported the general epidemiology data for bladder cancer, while data on urothelial carcinoma specifically were not available.5Despite chemotherapy has remained a key role in the treatment option for Chinese la/mUC patients for decades, novel treatments of ICIs, targeted therapy, and ADCs are rapidly evolving, with a handful of agents that demonstrated clinical promise have obtained approval. Beyond the established standards integrated in multiple guidelines, standardization of treatments for la/mUC patients might be a solution to improve the treatment effectiveness and advance the current treatment landscape. Furthermore, pioneering large research in real‐world setting is encouraged given the limited real‐world evidence in la/mUC patients, and particularly among Chinese population. With the heterogenous performance of gene alterations across different patients, future study could be promoted to better understand the molecular biomarkers and pathways in la/mUC patients, and further explore potential precise therapies for specific molecular subtypes.Wang He: Conceptualization (lead); methodology (lead); supervision ; writing – original draft ; writing – review and editing (lead). Changhao Chen: Formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft . Tianxin Lin: Supervision ; writing – original draft ; writing – review and editing . Qian Xu: Investigation ; validation ; writing – original draft ; writing – review and editing . Chong Ye: Data curation (lead); formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; writing – original draft . Jieyi Du: Funding acquisition ; project administration ; resources ; writing – original draft . Jian Huang: Conceptualization ; project administration ; supervision (lead); writing – review and editing .This work was supported by Xi'an Janssen Pharmaceutical Ltd. The authors were responsible for all content and editorial decisions, and received no honoraria related to the development of this publication.Qian Xu, Chong Ye, and Jieyi Du were employees of Xi'An Janssen Pharmaceutical Ltd during the study. Other authors declare no competing interests.Not applicable.Not applicable.Not applicable.Not applicable.Table S1‐S11Click here for additional data file.


ER+HER2+ breast cancer requires most types of systemic therapies perioperatively. However, treatment resistance is often experienced. The current study investigated the predictive and prognostic value of intratumoral heterogeneity and conventional clinicopathological factors in patients with ER+HER2+ breast cancer.This research included two patient cohorts with ER+HER2+ breast cancer. Cohort A included patients who underwent surgery without neoadjuvant chemotherapy (NAC). Cohort B comprised patients who received NAC followed by surgery. Intratumoral heterogeneity was assessed via ER and HER2 double staining, and the number of cells stained with different patterns of ER and HER2 was counted.p = 0.0013). Low ER and PgR Allred scores indicated better pCR rates than high scores . Multivariate analysis showed that the ER Allred score and cell component of ER−HER2+ cells without HER2− cells were independent predictors of pCR .In total, 11 of 92 tumors in cohort A and four of 45 tumors in cohort B consisted exclusively of double‐positive (ER+ and HER2+) cells (homogeneous). The rest had different combinations of cells (heterogeneous). The pathological complete response (pCR) rates differed based on tumoral cell components but not intratumoral heterogeneity. The pCR rate of tumors with ER−HER2+ cells but without HER2− cells was higher than that of others . The prognostic value of combined posttreatment Ki67 and pCR was superior to that of either alone. Combined pCR and posttreatment Ki67 had an independent prognostic value for DFS and OS .In cohort B, posttreatment Ki67, but not pCR, was a prognostic factor of DFS and OS (In ER+HER2+ breast cancer, the presence of ER−HER2+ cells without HER2− cells was independently associated with pCR. Combined posttreatment Ki67 and pCR can be more precise in predicting prognosis than pCR alone.  Therefore, they are useful in determining appropriate treatment strategies.In the NeoSphere and NeoALTTO trials, patients with HER2+ breast cancer received chemotherapy and anti‐HER2 therapy preoperatively. Results showed that the hormone receptor‐negative group had a higher pCR rate than the hormone receptor‐positive group.The intratumoral heterogeneity, in which the histology of cancer differs spatially and temporally within the same tumor, can cause treatment resistance.The current study assessed intratumoral heterogeneity by counting single cells via ER and HER2 double staining. Further, the predictive and prognostic value of intratumoral heterogeneity and other conventional clinicopathological factors in patients with ER+HER2+ breast cancer was investigated.22.1This is a retrospective observational study that included two patient cohorts with ER+HER2+ breast cancer. Cohort A included patients without neoadjuvant chemotherapy (NAC). The inclusion criteria were: unilateral invasive breast cancer that received surgical removal between January 2008 and December 2011 and ER+HER2+ breast cancer. The exclusion criteria were: preoperative systemic therapy administered and incisional biopsy performed at other hospitals. By reassessment of surgical specimen, no invasive cancer remaining, ER‐negative cancer or HER2‐negative cancer were also excluded. Cohort B comprised patients who received NAC. The inclusion criteria were: primary breast cancer that received surgery between January 2009 and December 2013, stage I‐III, HER2‐positive breast cancer, and NAC administered. The exclusion criteria were: needle biopsy performed at other hospitals and ER‐negative breast cancer.2.2Patients with ER+HER2+ breast cancer were treated based on the National Comprehensive Cancer Network guidelines Table .242, epirubicin 100 mg/m2, and 5‐fuorouracil 500 mg/m2, q3w) or six cycles of CAF , followed by four cycles of tri‐weekly docetaxel at a dose of 75 mg/m2 or 12 cycles of weekly paclitaxel at a dose of 80 mg/m2. Trastuzumab was concurrently added to the taxane regimen. In adjuvant therapy, four cycles of AC  or four cycles of AC, followed by taxane, were used.Neoadjuvant systemic therapy regimens comprised four cycles of CEF .The Kaplan–Meier curves were obtained, and the log‐rank test was applied to compare the survival distributions of two populations. 2.6This study was approved by the institutional review board and the ethical committee of the Japanese Foundation for Cancer Research (IRB‐2018‐1100). Patients' informed consent was waived by the ethical committee since this study was a retrospective investigation.33.1In total, 2754 patients had unilateral invasive breast cancer and received surgery between January 2008 and December 2011, of whom 165 had ER + HER2+ breast cancer. Out of these patients, 61 patients with preoperative systemic therapy, two patients who underwent incisional biopsy at other hospitals, three patients without evaluable invasive lesion in the surgical specimen, and three and four patients with ER− and HER2− cancer, respectively, on pathological reassessment were excluded. Hence, 92 patients were included in cohort A Figure . For cohIntratumoral heterogeneity was assessed by examining the ER and HER2 status of a single cell in each specimen Figure . Two 2.. Meanwhi3.2The predictive value of intratumoral heterogeneity and the other clinicopathological factors of pCR was examined in cohort B. First, the predictive value of intratumoral heterogeneity for pCR was assessed. Because the statistical power was too low due to a small number in the homogeneous group, the statistical analysis comparing between homogeneous and non‐homogeneous groups was not performed but no clear association was observed between intratumoral heterogeneity and pCR. Next, we investigated the predictive value of each cell component according to ER and HER2 status Table . The prep < 0.05 in the univariate analysis were included in the multivariate analysis. In model B, pathological stage and HER2 protein expression were further included to adjust for pathological stage, PgR status, and HER2 expression, which were associated with cell component in cohort B . In model B, cell component of ER−HER2+ cells without HER2− cells were an independent predictor of pCR even after adjusting for pathological stage, PgR status, and HER2 protein expression (p = 0.0098).Next, the association between conventional clinicopathological factors, including ER, PgR, HER2, and nuclear grade (NG), and pCR was examined. The cutoff values of ER and PgR Allred scores were set at 7 and 5, respectively, based on the Youden index via receiver operating characteristic curve analysis. As shown in Table  B Table . Logisti3.3p = 0.0036 and 0.0026, respectively; Figure p < 0.1 in the univariate analyses were included in the multivariate analysis in which ccombined pCR and posttreatment Ki67, but not posttreatment Ki67 alone, was included. Results showed that combined pCR and posttreatment Ki67 had an independent prognostic value for DFS and OS .The prognostic values of clinicopathological factors in both cohorts were analyzed. In cohort A, none of the factors, including pT, pN, ER, PgR, HER2, NG, and cell component, had a prognostic significance on DFS or OS Table . Because4The current study examined intratumoral heterogeneity via ER and HER2 double staining to simultaneously validate the expression of ER and HER2. Results showed that cell component, rather than intratumoral heterogeneity, affected response to NAC. Further, the presence of ER−HER2+ cells and the absence of HER2− cells indicated good pathological response even after adjusting for factors associated with cell component, which included pathological stage, PgR status, and HER2 protein expression. This result might be understandable considering that ER− tumors have higher pCR rates than ER+ tumors among patients with HER2+ breast cancers in clinical trialsPrevious studies have reported an association between intratumoral heterogeneity and treatment response,In this study, pCR was not a prognostic factor of ER+HER2+ breast cancer, and posttreatment Ki67 was associated with DFS and OS. The prognostic impact of pCR differs based on breast cancer subtype. A meta‐analysis of HER2+ breast cancer showed that the difference in survival between patients with pCR and those without was greater in hormone receptor‐negative breast cancer than in hormone receptor‐positive breast cancer .The current study had several limitations. First, the sample size, particularly in cohort B, was small. Thus, our results, including those of the prognostic analyses, should be interpreted with caution. Second, the assessment of cell components was not simple, and hundreds of single cells should be counted cautiously. However, artificial intelligence has been making a significant advancement in image analyses and can be useful for the assessment of cell components in ER+HER2+ breast cancer.5In ER+HER2+ breast cancer, cell component influenced treatment response. That is, the presence of ER−HER2+ cells and the absence of HER2− cells were associated with a good pCR rate. Furthermore, combined posttreatment Ki67 and pCR can be more precise in predicting prognosis than pCR alone. However, a larger prospective study should be conducted to validate these results.Natsuki Teruya: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation ; methodology ; resources ; software ; validation ; visualization ; writing – original draft . Hiroaki Inoue: Data curation ; formal analysis ; investigation ; methodology ; software . Rie Horii: Conceptualization ; data curation ; formal analysis ; methodology ; supervision . Futoshi Akiyama: Conceptualization ; data curation ; supervision . Takayuki Ueno: Conceptualization ; formal analysis ; writing – original draft ; writing – review and editing . Shinji Ohno: Resources ; supervision ; writing – review and editing . Shunji Takahashi: Conceptualization ; resources ; supervision ; writing – review and editing .This study was approved by the ethical committee of the Japanese Foundation for Cancer Research (IRB‐2018‐1100). Patients' informed consent was waived by the ethical committee since this study was a retrospective investigation.Data S1.Click here for additional data file.