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This study analysed the relationships between the main thromboelastography (TEG) parameters, the platelet (PLT) count and clinical bleeding in patients with blood diseases. We explored the threshold of the relevant parameters in the pathological condition of bleeding, aiming to scientifically guide clinical prophylactic PLT transfusion.In total, 268 patients with clear diagnoses of blood diseases and thrombocytopenia were enrolled and divided into five groups, A, B, C, D and E, corresponding to PLT counts of 0-10 × 10The maximum amplitude (MA) in groups A, B and C increased gradually, with a significant difference between each pair of these groups (P 0.05). In groups A, B, C, D and E, the corresponding MA at the time of bleeding was 43.5 mm, 39.6 mm, 38.0 mm, 35.2 mm and 50.5 mm, respectively, with statistically significant differences (P 0.05).The MA can be used as a reference indicator for preventive PLT transfusion to a certain extent. When the PLT count is within different ranges, the MA threshold for preventive PLT transfusion also differs. It is recommended that different PLT counts be correlated with different MA thresholds to guide clinical prophylactic PLT transfusion.

Relationship between CNA level and TMB in (A) MSK pan-cancer non-ICI treated cohort and (B) other three ICI-treated cohorts (MSK NSCLC, SKCM, and Dana-Farber ICI cohort). For each cancer type and each cohort, a plot is shown containing the CNA level versus tumor mutation burden. The spearman correlation coefficient and P value are shown.

Contradictory characteristics of elevated mutational burden and a “cold” tumor microenvironment (TME) coexist in liver kinase B1 (LKB1)–mutant non–small cell lung cancers (NSCLC). The molecular basis underlying this paradox and strategies tailored to these historically difficult to treat cancers are lacking. Here, by mapping the single-cell transcriptomic landscape of genetically engineered mouse models with Kras versus Kras/Lkb1-driven lung tumors, we detected impaired tumor-intrinsic IFNγ signaling in Kras/Lkb1-driven tumors that explains the inert immune context. Mechanistic analysis showed that mutant LKB1 led to deficiency in the DNA damage repair process and abnormally activated PARP1. Hyperactivated PARP1 attenuated the IFNγ pathway by physically interacting with and enhancing the poly(ADP-ribosyl)ation of STAT1, compromising its phosphorylation and activation. Abrogation of the PARP1-driven program triggered synthetic lethality in NSCLC on the basis of the LKB1 mutation–mediated DNA repair defect, while also restoring phosphorylated STAT1 to favor an immunologically “hot” TME. Accordingly, PARP1 inhibition restored the disrupted IFNγ signaling and thus mounted an adaptive immune response to synergize with PD-1 blockade in multiple LKB1-deficient murine tumor models. Overall, this study reveals an unexplored interplay between the DNA repair process and adaptive immune response, providing a molecular basis for dual PARP1 and PD-1 inhibition in treating LKB1-mutant NSCLC.Significance:Targeting PARP exerts dual effects to overcome LKB1 loss–driven immunotherapy resistance through triggering DNA damage and adaptive immunity, providing a rationale for dual PARP and PD-1 inhibition in treating LKB1-mutant lung cancers.

Demographic and clinicopathologic characteristics of three cohorts treated with immune checkpoint inhibitors.

Sequences for qPCR primers.

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient cells. (a) A549 lung cancer cells were transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) followed by treatment with olaparib (5μM) for 24 hours. The expression of pSTAT1 and PARP1 was analyzed by western blotting. (b-d) LLC1-shLkb1 and LLC1-shCtrl cells were pre-treated with olaparib (5μmol/L) for 6 hours and then stimulated with IFNγ for another 24 hours. Total pSTAT1 and STAT1 were quantified using western blot (b) and cell-surface PD-L1 was measured by flow cytometry (c). Q-PCR analysis was used to detect the expression of CXCL9 and PD-L1 (d). (e, f) The three groups of A549 cells were treated with IFNγ or IFNγ/olaparib and then refreshed the culture medium. The ratio of CD8+ T (effector cells) to A549 cells (target cell) ratio was 5:1. After 6 hours of incubation, QT-PCR analysis was used to detect the expression of indicated molecules of CD8+ T cells. (g) CD8+ T cells were treated with PBS or olaparib (5μmol/L) for 24 hours, and then the cells were collected for quantification of GZMB, PRF1, TNF, and IFNγ. CD8a was selected as the reference gene. (h) LLC-LV-Ctrl cells were treated with IFNγ/anti-PD-1 antibodies (20 μg/mL) in the absence or presence of olaparib (5 µM) or BMDCs (Tumor cells: DCs= 1:1) for 24 hours. Flow cytometry assay was used for apoptosis analysis of tumor cells. The right graph showed the quantification of tumor cell apoptosis. The data are presented as the mean ± SD of triplicate experiments. Statistical significance was determined by Student's t-test; ns, not significant; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

LKB1-interacting proteins identified by LC-MS/MS analysis.

Supplementary figure legends

Purpose:Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu.Experimental Design:Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy.Results:TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors.Conclusions:The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

Bo-Wen Jiang,1,2,* Xue Ji,1,2,* Zhong-Qing Lyu,3 Bing Liang,1,2 Jian-Hang Li,3 Ling-Wei Zhu,1,2 Xue-Jun Guo,1,2 Jun Liu,1,2 Yang Sun,1,2 Yan-Jing Liu3 1Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, People’s Republic of China; 2Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun, Jilin, People’s Republic of China; 3Third Affiliated Clinical Hospital to Changchun University of Chinese Medicine, Changchun, Jilin, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yang Sun; Yan-Jing Liu, Tel +86 431-86986933, Email sunyang10@hotmail.com; 214953156@qq.comPurpose: To elucidate the contribution of a transferable plasmid harboring the blaNDM-1 gene in an Escherichia coli clinical isolate to the spread of resistance determinants.Methods: Nine extended-spectrum β-lactamase-producing E. coli were collected from diarrhea samples from a pediatric patient and genetic linkage was investigated through enterobacteriaceae repetitive intragenic consensus polymerase chain reaction (PCR). Bacterial species were identified by 16s rRNA sequencing, susceptibility testing with the use of a BD PhoenixTM-100 Automated Microbiology System, and assessment of virulence genes by PCR. The transferability of blaNDM-1 in E. coli strain TCM3e1 was confirmed by conjugation experiments. Complete sequencing of E. coli strain TCM3e1 was determined with the PacBio and Illumina NovaSeq platforms and the characteristics were analyzed with bioinformatics software.Results: The results showed that all nine E. coli strains were the same clone. E. coli strain TCM3e1 was resistant to 12 antimicrobial agents and carried the virulence gene EAST-1. Conjugation transfer analysis showed that blaNDM-1 was carried on a self-transmissible plasmid. Two copies of the blaNDM-1 gene were present on an IncC plasmid and some resistance genes with two or three copies were located downstream of the blaNDM-1 gene and formed a tandem repeat fragment (blaDNM-1-bleo-sul1- aadA17- dfrA12).Conclusion: A transmissible plasmid harboring two copies of the blaNDM-1 gene, including clonal dispersions of the blaNDM-1 gene, was identified in clinical isolates. These findings emphasized the necessity of surveillance of the plasmid-borne blaNDM-1 to prevent dissemination.Keywords: diarrheagenic Escherichia coli, multi-drug-resistant, New Delhi metallo-β-lactamase, blaDNM-1 gene

Contradictory characteristics of elevated mutational burden and a “cold” tumor microenvironment (TME) coexist in liver kinase B1 (LKB1)–mutant non–small cell lung cancers (NSCLC). The molecular basis underlying this paradox and strategies tailored to these historically difficult to treat cancers are lacking. Here, by mapping the single-cell transcriptomic landscape of genetically engineered mouse models with Kras versus Kras/Lkb1-driven lung tumors, we detected impaired tumor-intrinsic IFNγ signaling in Kras/Lkb1-driven tumors that explains the inert immune context. Mechanistic analysis showed that mutant LKB1 led to deficiency in the DNA damage repair process and abnormally activated PARP1. Hyperactivated PARP1 attenuated the IFNγ pathway by physically interacting with and enhancing the poly(ADP-ribosyl)ation of STAT1, compromising its phosphorylation and activation. Abrogation of the PARP1-driven program triggered synthetic lethality in NSCLC on the basis of the LKB1 mutation–mediated DNA repair defect, while also restoring phosphorylated STAT1 to favor an immunologically “hot” TME. Accordingly, PARP1 inhibition restored the disrupted IFNγ signaling and thus mounted an adaptive immune response to synergize with PD-1 blockade in multiple LKB1-deficient murine tumor models. Overall, this study reveals an unexplored interplay between the DNA repair process and adaptive immune response, providing a molecular basis for dual PARP1 and PD-1 inhibition in treating LKB1-mutant NSCLC.Significance:Targeting PARP exerts dual effects to overcome LKB1 loss–driven immunotherapy resistance through triggering DNA damage and adaptive immunity, providing a rationale for dual PARP and PD-1 inhibition in treating LKB1-mutant lung cancers.

Yan-hui Wei,1 Yu-zhuo He,2 Xiao-yan Guo,1 Xiao-yan Lin,2 Hong-bin Zhu,2 Xue-jun Guo2 1Department of Graduate School, Xinxiang Medical University, Xinxiang, Henan Province, 453003, People’s Republic of China; 2Department of Hematology, Puyang Oilfield General Hospital, Puyang, Henan Province, 457000, People’s Republic of ChinaCorrespondence: Xue-jun GuoDepartment of Hematology, Puyang Oilfield General Hospital, Puyang, Henan Province, 457000, People’s Republic of ChinaTel/Fax +86-13903930612Email pygxj@163.comObjective: This study aimed to determine the incidence of iron-deficiency anemia (IDA) complicated by splenomegaly in our hospital over the past 6 years and to analyze the possible causes of this result.Methods: This is a retrospective study. In total, 668 patients with IDA who were hospitalized in the hematology department of our hospital from 2013 to 2019 were selected as the research subjects and included in the IDA group, and 3201 patients who underwent outpatient physical examinations in our hospital during the same period were included in the control group. The incidences of splenomegaly in the IDA and control groups were calculated, and the difference was analyzed by means of statistical methods.Results: Among the 668 IDA patients, 46 (6.9%) had splenomegaly, and among the 3201 patients in the control group, 21 had splenomegaly (0.7%). The incidence of splenomegaly was significantly higher in the IDA group than in the control group, and the severity of anemia in the IDA group was associated with the occurrence of splenomegaly. Specifically, the incidence of splenomegaly was 12.4% among patients with severe anemia and as high as 50% among patients with extremely severe anemia.Conclusion: IDA is correlated with the incidence of splenomegaly, and the incidence of splenomegaly significantly increases as the severity of IDA increases. This is considered to be caused by extramedullary hematopoiesis.Keywords: iron-deficiency anemia, splenomegaly, extramedullary hematopoiesis, cytokines

Microsatellite instability (MSI) has been approved as a pan-cancer biomarker for immune checkpoint blockade (ICB) therapy. However, current MSI identification methods are not available for all patients. We proposed an ensemble multiple instance deep learning model to predict microsatellite status based on histopathology images, and interpreted the pathomics-based model with multi-omics correlation. Methods: Two cohorts of patients were collected, including 429 from The Cancer Genome Atlas (TCGA-COAD) and 785 from an Asian colorectal cancer (CRC) cohort (Asian-CRC). We established the pathomics model, named Ensembled Patch Likelihood Aggregation (EPLA), based on two consecutive stages: patch-level prediction and WSI-level prediction. The initial model was developed and validated in TCGA-COAD, and then generalized in Asian-CRC through transfer learning. The pathological signatures extracted from the model were analyzed with genomic and transcriptomic profiles for model interpretation. Results: The EPLA model achieved an area-under-the-curve (AUC) of 0.8848 (95% CI: 0.8185-0.9512) in the TCGA-COAD test set and an AUC of 0.8504 (95% CI: 0.7591-0.9323) in the external validation set Asian-CRC after transfer learning. Notably, EPLA captured the relationship between pathological phenotype of poor differentiation and MSI (P < 0.001). Furthermore, the five pathological imaging signatures identified from the EPLA model were associated with mutation burden and DNA damage repair related genotype in the genomic profiles, and antitumor immunity activated pathway in the transcriptomic profiles. Conclusions: Our pathomics-based deep learning model can effectively predict MSI from histopathology images and is transferable to a new patient cohort. The interpretability of our model by association with pathological, genomic and transcriptomic phenotypes lays the foundation for prospective clinical trials of the application of this artificial intelligence (AI) platform in ICB therapy.

Background Organ-specific metastatic context has not been incorporated into the clinical practice of guiding programmed death-(ligand) 1 [PD-(L)1] blockade, due to a lack of understanding of its predictive versus prognostic value. We aim at delineating and then incorporating both the predictive and prognostic effects of the metastatic-organ landscape to dissect PD-(L)1 blockade efficacy in non-small cell lung cancer (NSCLC). Methods A total of 2062 NSCLC patients from a double-arm randomized trial (OAK), two immunotherapy trials (FIR, BIRCH), and a real-world cohort (NFyy) were included. The metastatic organs were stratified into two categories based on their treatment-dependent predictive significance versus treatment-independent prognosis. A metastasis-based scoring system (METscore) was developed and validated for guiding PD-(L)1 blockade in clinical trials and real-world practice. Results Patients harboring various organ-specific metastases presented significantly different responses to immunotherapy, and those with brain and adrenal gland metastases survived longer than others [overall survival (OS), p = 0.0105; progression-free survival (PFS), p = 0.0167]. In contrast, survival outcomes were similar in chemotherapy-treated patients regardless of metastatic sites (OS, p = 0.3742; PFS, p = 0.8242). Intriguingly, the immunotherapeutic predictive significance of the metastatic-organ landscape was specifically presented in PD-L1-positive populations (PD-L1 > 1%). Among them, a paradoxical coexistence of a favorable predictive effect coupled with an unfavorable prognostic effect was observed in metastases to adrenal glands, brain, and liver (category I organs), whereas metastases to bone, pleura, pleural effusion, and mediastinum yielded consistent unfavorable predictive and prognostic effects (category II organs). METscore was capable of integrating both predictive and prognostic effects of the entire landscape and dissected OS outcome of NSCLC patients received PD-(L)1 blockade (p < 0.0001) but not chemotherapy (p = 0.0805) in the OAK training cohort. Meanwhile, general performance of METscore was first validated in FIR (p = 0.0350) and BIRCH (p < 0.0001), and then in the real-world NFyy cohort (p = 0.0181). Notably, METscore was also applicable to patients received PD-(L)1 blockade as first-line treatment both in the clinical trials (OS, p = 0.0087; PFS, p = 0.0290) and in the real-world practice (OS, p = 0.0182; PFS, p = 0.0045). Conclusions Organ-specific metastatic landscape served as a potential predictor of immunotherapy, and METscore might enable noninvasive forecast of PD-(L)1 blockade efficacy using baseline radiologic assessments in advanced NSCLC.

21 In August 2018, hundreds of migratory birds died in the area of Dali Nouer Lake, Chifeng City, 22 China. We collected the remains of dead birds along with water and aquatic plants from the birds’ 23 environment. The bacterial communities of all samples were profiled by high-throughput 24 sequencing of the V3–V4 hypervariable region of the 16S rRNA amplicon. At the genus level, 25 Bacteroides, Clostridium, Plesiomonas, Vibrio, Fusobacterium, and Aeromonas were the 26 dominant genera in dead birds, the lake water, and aquatic plants in 2018. However, the relative 27 abundances of these bacterial genera were significantly reduced compared with the levels obtained 28 from healthy migratory bird feces, lake water, and aquatic plants from the same place and time 29 period in 2019. Combined with environmental factors such as the changes in salt content and pH, 30 the invasion and reproduction of those pathogens may have promoted the decline and death of the 31 birds. 32 33

Introduction Studies of the effects of stem cell therapy on type 2 diabetes mellitus (T2DM) have not reached consistent results. Our meta-analysis aimed to systematically evaluate the efficacy of autologous bone marrow-derived stem cells (ABM-MNCs) on T2DM. Methods We systematically searched PubMed, EMBASE, Web of Science, and the Cochrane Library for studies published between 1980 and May 2018. Two researchers screened the literature independently following the inclusion and exclusion criteria. Meta-analysis of the pooled standard mean difference (SMD) with 95% confidence interval (CI) was calculated based on either a fixed- or random-effects model. Results We identified six studies with 206 participants investigating the effects of autologous bone marrow stem cell therapy on T2DM after screening 102 studies found after the initial search. According to the pooled estimates, compared with the control group, after 12-month follow-up the ABM-MNC therapy group had a lower level of HbA1c (MD, − 1.18; 95% CI, − 1.40 to 0.95) and lower required insulin dose (MD, − 2.05; 95% CI, − 3.55 to − 0.55). HbA1c decreased after ABM-MNC therapy compared with before (12 months: MD, − 1.22; 95% CI, − 1.43 to − 1.0). We also observed a significant decrease in insulin requirement after 3-, 6-, 9-, and 12-month follow-up in the ABM-MNC group, respectively. Conclusion Autologous stem cell therapy showed a beneficial effect on T2DM.

Objectives: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is heterogeneous in clinical practice. Materials and Methods: We retrospectively assessed the predicitive effect of the primary and metastatic lesion spectrum (baseline sum of the longest diameters [SLD], number of metastatic sites and specific organ metastases) on the efficacy of atezolizumab over docetaxel in OAK and POPLAR trial cohorts. A decision model, termed DSO (Diameter-Site-Organ), based on the spectrum was developed and validated for guiding ICB. Results: Higher SLD (>38 mm) and more metastatic sites (≥2) were characterized with pronounced overall survival (OS) benefits from atezolizumab versus docetaxel. Specifically, adrenal gland and brain metastases were identified as favorable predictors of atezolizumab treatment. The DSO model was developed in the discovery cohort to integrate the directive effect of the primary and metastatic lesion spectrum. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a significantly prolonged OS than docetaxel, whereas OS was generally similar between two treatments in patients with DSO score ≤ 0. Equivalent findings were also seen in the internal and external validation cohorts. Conclusions: The response to anti-PD-L1 therapy among patients varied with the primary and metastatic lesion spectrum. The DSO-based system might provide promising medication guidance for ICB treatment in NSCLC patients.

Background: Tuberculosis pleural effusion patients always have elevated D-dimer levels. How D-dimer performance in predicting pulmonary embolism in TPE population is unclear. The aim of this study was to assess the diagnostic performance of D-dimer for pulmonary embolism in TPE population and explore the potential mechanism of PE in TPE patients.Methods: We retrospectively analyzed patients who were admitted to Xinhua hospital and Weifang Respiratory Disease Hospital with a final diagnosis of TPE between March 2014 and January 2020. Data including demographics, history, symptoms, D-dimer, C-reactive protein (CRP), white blood cell (WBC) and erythrocyte sedimentation rate (ESR) as well as the cytokines such as IL-6, IL-8, TNF-α, IL-2 receptor of the first blood test, imaging results including computed tomography pulmonary angiography (CTPA) and compression ultrasound (CUS) with Doppler were collected and analyzed. Results: 248 patients (male=170, female=78) at the age of 43±20.6 years were final included in this study. Elevated D-dimer (≥0.5mg/L) was detected in 186/248 (75%) patients. 150 patients received CTPA examination. PE was diagnosed in 29/150 (19.3%) patients. Among the TPE population, PE patients had significantly higher DD level than that of non-PE patients (2.26±0.96 mg/L VS 1.06±0.73 mg/L) (P＜0.05). An optimized cut off value for D-dimer in predicting PE in TPE was 1.18 mg/L, with a sensitivity of 89.7% and a specificity of 77.8 % ( area under curve was 0.893; 95% CI: 0.839-0.947; P＜0.01). PE patients had lower median WBC and IL-8 values (5.14Ⅹ109/L VS 6.1Ⅹ109/L, P＜0.05 ; 30.2 pg/ml VS 89.7 pg/ml, P＜0.05) but higher median IL-2 receptor value (1964.8 pg/ml VS 961.2 pg/ml, P＜0.01) than that of non-PE patients.Conclusions: D-dimer is still an objective biomarker to predict PE in TPE patients. In order to avoid unnecessary radiological test, the cut off value of D-dimer in TPE patients should be set at 1.18 mg/L. In addition, the imbalance of prothrombotic and antithrombotic cytokines may partly attribute to the formation of pulmonary embolus in TPE patients.

Migratory birds are recently recognized as Vibrio disease vectors, but may be widespread transporters of Vibrio strains. We isolated Vibrio cholerae (V. cholerae) and Vibrio metschnikovii (V. metschnikovii) strains from migratory bird epidemic samples from 2017 to 2018 and isolated V. metschnikovii from migratory bird feces in 2019 from bird samples taken from the Inner Mongolia autonomous region of China. To investigate the evolution of these two Vibrio species, we sequenced the genomes of 40 V. cholerae strains and 34 V. metschnikovii strains isolated from the bird samples and compared these genomes with reference strain genomes. The pan-genome of all V. cholerae and V. metschnikovii genomes was large, with strains exhibiting considerable individual differences. A total of 2,130 and 1,352 core genes were identified in the V. cholerae and V. metschnikovii genomes, respectively, while dispensable genes accounted for 16,180 and 9,178 of all genes for the two strains, respectively. All V. cholerae strains isolated from the migratory birds that encoded T6SS and hlyA were non-O1/O139 serotypes without the ability to produce CTX. These strains also lacked the ability to produce the TCP fimbriae nor the extracellular matrix protein RbmA and could not metabolize trimetlylamine oxide (TMAO). Thus, these characteristics render them unlikely to be pandemic-inducing strains. However, a V. metschnikovii isolate encoding the complete T6SS system was isolated for the first time. These data provide new molecular insights into the diversity of V. cholerae and V. metschnikovii isolates recovered from migratory birds.

Background: In August 2018, a large number of migratory birds died in Chifeng, Neimeng. We were entrusted by local animal disease prevention and control center to collect the migratory bird epidemic materials and their living environmental water, and in 2019, we collect the local migratory bird stool and their living environmental water again. The bacterial communities in migratory bird epidemic materials, water and aquatic plants are profifiled by high-throughput sequencing of the V3–V4 hypervariable region of 16S rDNA gene.Results: We found that the dominant phylum between migratory bird epidemic materials, water and aquatic plant were Proteobacteia, Bacteroidetes, Firmicutes, Fusobacteria and Verrucomicrobia in 2018. One year later, we found that the dominant phylum between migratory bird stool, water and aquatic plant were Proteobacteia, Firmicutes, Cyanobacteria, Bacteroidetes. The relative abundance among bacterial phylum notably differed between two years. The relative abundance of Fusobacteria and Verrucomicrobia were higher in samples in 2018, while that of Cyanobacteria was higher in water, 2019. The relative abundance of Fusobacteria in migratory bird epidemic materials began to decline in the later period over time. At genus level, the relative abundance of Vibrio, Clostridium and other patnogenic bacteria decreased markedly and disappeared in 2019. The salt content and pH show a downward trend.Conclusions: Differences in diet and geographical location can lead to diversification in migratory bird intestinal flora. It is necessary to pay attention to diversification in intestinal flora of migratory birds, especially the abundance of vibrio in intestine. The overall structure of intestinal flora and relative abundance changes of various species are showed intuitively by 16S rDNA amplicon. But comparing to PCR which using specific primers, the accuracy and sensitivity are relatively poor. In the case of purposeful detection, it can be used in combination with both 16S rDNA amplicon and PCR which using specific primers. They are more accurately, because of supplement each other.

Background Lymphoma is a common haematological malignancy with many subtypes and considerable heterogeneity. Traditional treatments include chemotherapy, radiotherapy and surgery. Patients with relapsed, refractory or advanced stage lymphoma have a dismal prognosis. In recent years, chimeric antigen receptors (CARs) have been recognized as powerful tools that redirect antigen-specific T cells independent of human lymphocyte antigen (HLA) restriction and specifically kill tumour cells. Satisfactory results with CAR-based treatments have been achieved in relapsed/refractory B cell leukaemia/lymphoma. Our centre explored the strategy of subcutaneous injections combined with intravenous drip to overcome certain issues. Case presentation A patient with stage IV refractory and relapsed diffuse large B cell lymphoma was treated with regional and intravenous CAR-T cells. During the observation period, the temperature of the skin at the abdominal wall mass was slightly elevated, and tolerable pain in the injection area was reported. Imaging showed regional liquefactive necrosis. No specific medical intervention was applied. After the sequential administration of ibrutinib and venetoclax, the abdominal wall mass significantly decreased in size. Conclusion The regional injection of CAR-T cells might be safe and feasible for the treatment of regional lesions in patients with refractory and relapsed advanced lymphoma.

BackgroundMicrosatellite instability (MSI) is a negative prognostic factor for colorectal cancer (CRC) and can be used as a predictor of success for immunotherapy in pan-cancer. However, current MSI identification methods are not available for all patients. We propose an ensemble multiple instance learning (MIL)-based deep learning model to predict MSI status directly from histopathology images.DesignTwo cohorts of patients were collected, including 429 from The Cancer Genome Atlas (TCGA-COAD) and 785 from a self-collected Asian data set (Asian-CRC). The initial model was developed and validated in TCGA-COAD, and then generalized in Asian-CRC through transfer learning. The pathological signatures extracted from the model are associated with genotypes for model interpretation.ResultsA model called Ensembled Patch Likelihood Aggregation (EPLA) was developed in the TCGA-COAD training set based on two consecutive stages: patch-level prediction and WSI-level prediction. The EPLA model achieved an area-under-the -curve (AUC) of 0.8848 in the TCGA-COAD test set, which outperformed the state-of-the-art approach, and an AUC of 0.8504 in the Asian-CRC after transfer learning. Furthermore, the five pathological imaging signatures identified using the model are associated with genomic and transcriptomic profiles, which makes the MIL model interpretable. Results show that our model recognizes pathological signatures related to mutation burden, DNA repair pathways, and immunity.ConclusionOur MIL-based deep learning model can effectively predict MSI from histopathology images and are transferable to a new patient cohort. The interpretability of our model by association with genomic and transcriptomic biomarkers lays the foundation for prospective clinical research.

Background: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is still heterogeneous in clinical practice. Methods: We retrospectively assessed the impact of baseline sum of the longest diameters (SLD), number of metastatic sites and specific organ metastases on the efficacy of atezolizumab versus docetaxel in the pooled population from OAK and POPLAR studies. An assistant decision model based on the machine-learning method, incorporating these indicators, termed DSO (Diameter-Site-Organ), was developed and validated in OAK and POPLAR cohorts. Findings: Higher SLD (> 38mm) and more metastatic sites (≥ 2) were characterized with pronounced OS benefits from atezolizumab versus docetaxel. Specifically, brain and adrenal gland metastases were identified as favorable predictors of atezolizumab treatment. The DSO model for guiding second-line treatment was developed based on SLD and metastatic sites and organs in the discovery cohort. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a prolonged OS than docetaxel (hazard ratio [HR] 0.67, 95% CI 0.54-0.84; P = 0.0003), whereas OS was generally similar between two treatments in patients with DSO score ≤ 0 (HR 1.46, 95% CI 0.89-2.39; P = 0.1346). Equivalent findings were seen in the internal and external validation cohorts. Interpretation: Our study for the first time revealed that patients with higher tumor burden were more suitable for ICB compared with standard chemotherapy. More importantly, the integrative DSO decision model might provide promising medication guidance for second-line ICB treatment in unselected NSCLC patients. Funding: This study was supported by the National Natural Science Foundation for YoungScientists of China (Grant No. 81802863 and 81902353), the Natural Science Foundation of Guangdong Province (Grant No. 2018030310285), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (Grant No. 2017J003). Conflict of Interest: The authors have no actual or potential conflicts of interest to declare. Ethical Approval: The study was approved by theInstitutional Ethical Review Boards of Nanfang Hospital. All patients enrolled in OAK and POPLAR provided signed informed consent in accordance with the protocols of the corresponding studies.

To analyze the risk factors affecting the chemotherapy-related infections in patients with acute lympho-blastic leukemia (ALL).The clinical data of 102 patients with ALL from January 2014 to December 2018 were collected and retrospectively studies. The risk factors of chemotherapy-related infections were analyzed by univa-riate and multivariate logistic regression.A total of 386 courses of chemotherapy were completed, out of which the infection occurred in 201 course, with the infection rate of 52.07%, identified infection number was 215 case-times, including perianal infection of 13.95% (30/215), oral infection of 13.49% (29/215), blood flow infection of 1721% (37/215), lower respiratory tract infection of 37.21% (80/215), urinary infection of 3.26% (7/215), skin infection of 3.72% (8/215), digestive and intra abdominal infection of 9.30% (20/215), and other infections of 1.86 (4/215). Totally 88 strains of pathogenic bacteria were detected, including 29 Gram-positive bacteria (32.95%), 52 Gram-negative bacteria (59.09%) and 7 fungi (7.95%). Gram-positive bacteria mainly were Staphylococcus haemolyticus and Enterococcus faecium, susceptible to tegacycline, vancomycin and linezolid; Gram-negative bacteria mainly were Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae, susceptible to tegacycline, amikacin, piperacillin/tazobactam and imipenem; Candida was the dominant fungus. Living in an ordinart ward, neutrophil defi-ciency for more than 7 days after chemotherapy and incomplete remission were independent risk factors of related infections during the induction chemotherapy in ALL inpatients, and hospitalization time also closely related with chemo-therapy-related infections in ALL inpatients (P0.05). Neutrophil deficiency for more than 7 days after chemotherapy was an independent risk factor of chemotherapy-related infections in ALL inpatients in the consolidation chemotherapy (P0.05).Patients with ALL are prone to chemotherapeutic-related infections, and those who lack neutrophils for more than 7 days after chemotherapy and who do not reach complete remission are more prone to infection. Living in laminar flow ward and reducing hospitalization stay can help reduce the incidence of infection.影响住院ALL患者化疗相关感染的危险因素分析.分析影响住院急性淋巴细胞白血病（ALL）患者化疗相关感染的危险因素.收集本院2014年1月至2018年12月102例住院ALL患者的临床资料，采用单因素和多因素Logistic回归分析影响患者化疗相关感染的危险因素.102例患者共完成化疗疗程386个，其中201个疗程发生感染，感染率为52.07%。明确感染部位215例次，其中肛周感染占13.95%（30/215），口腔感染占13.49%（29/215），血流感染占17.21%（37/215），下呼吸道感染感染占37.21%（80/215），泌尿系统感染占3.26%（7/215），皮肤感染占3.72%（8/215），消化系统与腹腔感染占9.30%（20/215），其它感染占1.86%（4/215）。共检出病原菌88株，其中革兰阳性菌29株（32.95%），革兰阴性菌52株（59.09%），真菌7株（7.95%）。革兰阳性菌中，主要为溶血葡萄球菌和屎肠球菌，对替加环素、万古霉素和利奈唑胺敏感；革兰阴性菌中，主要为铜绿假单胞菌、大肠埃希菌和肺炎克雷伯菌，对替加环素、阿米卡星、哌拉西林/他唑巴坦、亚胺培南等敏感；真菌以念珠菌属为主。住普通病房ALL患者、化疗后中性粒细胞缺乏时间超过7 d、未达完全缓解均是住院ALL患者诱导化疗期相关感染的独立危险因素，并且住院时间与住院ALL患者诱导化疗期相关感染存在密切关系（P0.05）；化疗后中性粒细胞缺乏时间超过7 d是住院ALL患者巩固化疗期相关感染的独立危险因素（P0.05）.住院ALL患者容易出现化疗相关感染，且化疗后中性粒细胞缺乏时间超过7 d、 未达完全缓解者更容易出现感染， 而住层流病房和减少住院时间有助于降低感染的发生率.

Chemotherapy and radiotherapy predominantly improve the clinical outcomes of patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC). Whether this superiority goes on when treated with immune checkpoint inhibitors is still unclear. This study sought to determine the predictive value and potential mechanisms of HPV status for the treatment of programmed cell death 1 (PD-1)/ligand 1(PD-L1) inhibitors. We conducted an integrated analysis of the relationships between HPV status and PD-L1, tumor mutation burden (TMB) and inflammation-related immune cells and molecules, based on the analysis of repository databases and resected HNSCC specimens. The pooled analysis of overall survival (OS) and objective response rate (ORR) suggested that HPV-positive patients benefited more from PD-1/PD-L1 inhibitors than HPV-negative patients (OS: hazard ratio (HR) = 0.71, p = 0.02; ORR: 21.9% vs 14.1%, odds ratio (OR) = 1.79, p = 0.01). Analysis of public databases and resected HNSCC specimens revealed that HPV status was independent of PD-L1 expression and TMB in HNSCC. However, HPV infection significantly increased T-cell infiltration, immune effector cell activation and the diversity of T-cell receptors. Notably, HPV-positivity correlated with increased immune cytolytic activity and a T-cell-inflamed gene expression profile. This work provides evidence that HPV status can be used to predict the effectiveness of PD-1 inhibitors in HNSCC, independently of PD-L1 expression and TMB, and probably results from an inflamed immune microenvironment induced by HPV infection.

Purpose: Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu. Experimental Design: Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy. Results: TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors. Conclusions: The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

Objective: Emerging evidence suggests that interleukin 37 (IL-37) plays an important role in the pathogenesis of several autoimmune diseases (ADs), but the correlations are still unclear. We conducted a meta-analysis to explore whether IL-37 gene (rs3811047) polymorphism was associated with susceptibility to multiple ADs in a Chinese population. Methods: Relevant studies were searched in the PubMed, Embase, Chinese National Knowledge Infrastructure, and Chinese Wangfang databases up to August 31, 2017. Odds ratio (OR) and its 95% confidence interval (95% CI) was used to estimate the strength of the association in different genetic models. The results of fixed or random models were adopted according to the heterogeneity. Publication bias and sensitive analysis were also performed to evaluate the reliability of results. Results: A total of 3161 patients and 4078 controls from 6 studies were included in this meta-analysis. Pooling all data together, a significant association between IL-37 gene (rs3811047 A/G) polymorphism and susceptibility to ADs in the Chinese population was found in all 4 genetic models (allelic model A vs G: OR = 0.73 95% CI = 0.67∼0.79; recessive model AA + AG vs GG: OR = 0.72, 95% CI = 0.65∼0.79; dominant model AA vs AG + GG: OR = 0.59, 95% CI = 0.45∼0.77; homozygous model AA vs GG: OR = 0.55, 95% CI = 0.42∼0.72). No heterogeneity and publication bias was detected in all models. Sensitive analysis indicated that all of the positive results are reliable. Conclusion: The IL- 37 (rs3811047) polymorphism contributes to the development of ADs in a Chinese population.

The development of chemoresistance may reduce the efficacy of chemotherapeutic drugs for treating hepatocellular carcinoma (HCC). In the present study, the effects of apigenin on intensifying the chemosensitivity of HCC cells and an HCC xenograft model in response to 5-fluorouracil (5-FU) were investigated. Sub-toxic concentrations of apigenin (4 μmol/L) significantly enhanced the cytotoxicity of 5-FU (100 μg/mL) in HCC cells. In vivo, combined treatment with apigenin (20 mg/kg, five times/week for 3 weeks) and 5-FU (20 mg/kg for 5 consecutive days) significantly inhibited the growth of HCC xenograft tumours. Annexin V-propidium iodide dual staining assays, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling assays and western blotting analysis were used to confirm the synergistic effects of apigenin and 5-FU on HCC apoptosis. Coincubation of HCC cells with apigenin and 5-FU increased levels of reactive oxygen species (ROS), which was followed by a decrease in the mitochondrial membrane potential (ΔΨm). In addition, combined triggered the mitochondrial apoptotic pathway, as indicated by decreased Bcl-2 expression and loss of ΔΨm, with significant activation of caspase 3 and poly(ADP-ribose) polymerase. The present study is the first to demonstrate that apigenin may potentiate the cytotoxicity of 5-FU in HCC via inhibition of ROS-mediated drug resistance and concurrent activation of the mitochondrial pathways of apoptosis.

Background Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. Methods Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. Results Five strains isolated from diseased mules were clustered together with patients’ isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. Conclusions It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0218-6) contains supplementary material, which is available to authorized users.

Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.

Rationale:Primary malignant melanoma of the lung (PMML) is an extremely rare neoplasm with a dismal prognosis. The diagnosis of PMML is very difficult and is based on several clinical, radiological, and histopathological criteria.Patient concerns:A 61-year-old women was admitted with a 2-mon

Background: The burden and severity of a cancer can be reflected by patterns of survival. Breast cancer prognosis between two countries with a different socioeconomic status and cultural beliefs may exhibit wide variation. This study aimed to describe survival in patients with breast cancer in China and Thailand in relation to demographic and clinical prognostic information. Materials and Methods: We compared the survival of 1,504 Chinese women in Yunnan province and 929 Thai women in Songkhla with breast cancer from 2006 to 2010. Descriptive prognostic comparisons between the Chinese and Thai women were performed by relative survival analysis. A Cox regression model was used to calculate the hazard ratios of death, taking into account the age, disease stage, period of diagnosis and country. Results: The overall 5-year survival proportion for patients diagnosed with breast cancer for Yunnan province (0.72) appeared slightly better than Songkhla (0.70) without statistical significance. Thai women diagnosed with distant and regional breast cancer had poorer survival than Chinese women. Disease stage was the most important determinant of survival from the results of Cox regression model. Conclusions: Breast cancer patients in Kunming had slightly greater five-year survival rate than patients in Songkhla. Both Chinese and Thai women need improvement in prognosis, which could conceivably be attained through increased public education and awareness regarding early detection and compliance to treatment protocols.

Objective: To analyse the microsatellite instability (MI) and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Methods: MI and LOH at chromosome 17 were checked in 35 cases of NSCLC tumor-normal paired tissues using four microsatellite markers TP53 (17pl3.1), THRA1 (17qll.2–12), D17S579 (17ql2–21) and D17S855 (17q21) by PCR based analysis. Mutations of P53 exons 5–8 were also tested using PCR-single strand conformation polymorphism (PCR-SSCP) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Results: 22 of 35 tumors (62.8%) displayed MI or LOH. 14 tumors (40.0%) exhibited MI, 11 tumors (31.4%) exhibited LOH, while 3 tumors (8.6%) exhibited MI and LOH concurrently. 23 tumors (65.7%) exhibited P53 gene mutations. The frequency of MI or LOH was obviously higher in the early-stage (stages I and II, 78.9%) than in the advanced-stage (stage III, 43.8%). However, the frequency of MI or LOH had no difference either between high-grade (75.0%) and low-grade (52.6%) differentiated NSCLC or between the tumors with P53 mutations (59.1%) and those without P53 mutations (69.2%). No relationship was observed between the presence of MI or LOH and the histologic subtype of NSCLC. Conclusion: The results suggest that MI and LOH at chromosome 17 may play a significant role in the development of NSCLC. The high frequency of MI or LOH in the early-stage tumors indicates that these genetic alterations could occur early during NSCLC development. Our data also show that the genetic alterations of microsatellite at chromosome 17 was not associated with P53 gene mutations.

To explore a more optimal therapy for intractable insomnia.Seven hundred cases of intractable insomnia that were in accordance with the criteria were randomly divided into an observation group (368 cases) and a control group (332 cases). The acupuncture prescription of regulating yin-yang and five viscera was applied in the observation group, where Dazhui (GV 14), Shenmai (BL 62), Guanyuan (CV 4), Zhaohai (KI 6), Geshu (BL 17), etc. were selected. The acupuncture prescription of tranquilizing mind was applied in the control group, where Baihui (GV 20), Sishencong (EX-HN 1), Anmian (Extra), Shenmen (HT 7), Sanyin jiao (SP 6) were selected. The treatment was given once a day, ten times of which made a session. After the treatment for 4 sessions, the clinical efficacy and Pittsburgh sleep quality index (PSQI) were compared between two groups.The total effective rate was 92.6% (338/365) in the observation group, which was superior to 73.1% (242/331) in the control group (P0.05). The PSQI score was obviously decreased in two groups after the treatment (both P0.05), in which the decreasing in the observation group was superior to that in the control group (P0.05).The acupuncture prescription of regulating yin-yang and five viscera has better effect for intractable insomnia, which could be considered as a more optimal therapy.

Bronchial anthracofibrosis (BAF) has been defined as a luminal narrowing associated with anthracotic pigmentation on bronchoscopy without a relevant history of pneumoconiosis or smoking. The aim of the study is to study the clinical features and imaging manifestations of BAF, and to promote the awareness of this disease.Between October 2006 and January 2012, 15 patients were diagnosed at our department as BAF that showed a narrowing or obliteration of lobar or segmental bronchi with anthracotic pigmentation in the overlying mucosa on bronchoscopy. The medical records including clinical features, imaging manifestations, electronic bronchoscopic findings, and pathological features were analysed, and the literature was reviewed.A total of 15 patients were analyzed; 13 were female (86.7%) and two were male (13.3%) and the age range was from 62 to 86 years with a mean age of 74.5 years. Three cases (20.0%) had a history of tuberculosis. The most common clinical symptoms of BAF were cough (100%), expectoration (73.3%), dyspnea (60.0%), and fever (46.7%). Twelve cases displayed mild to moderate obstructive ventilatory dysfunction. In the electronic bronchoscopic evaluation, the most common findings were black bronchial mucosal pigmentation, bronchial stenosis, bronchial occlusion, and bronchial mucosal folds. Pathological evaluation revealed chronic inflammation of the bronchial mucosa, submucosal carbon particle deposition, and mucosal or submucosal fibrosis. Chest CT scans showed that 15 patients had bronchial stenosis or obstruction (direct signs) with the right middle lobe being the most common site (11 cases, 73.3%). The indirect sign was mainly the presence of bronchial obstructive diseases (including secondary infection), represented by 11 cases of pulmonary consolidation (73.3%), seven cases of atelectasis (46.7%), and five cases of nodules (33.3%). The CT mediastinal window showed bronchial lymph node lesions, mediastinal lymph node calcification (12 cases, 80.0%), and enlargement of multiple mediastinal lymph nodes.The diagnosis of BAF was mainly based on bronchoscopic evaluation. Its pathogenesis is currently unclear, although it may be related to tuberculosis or bio-fuel inhalation. The diagnosis of BAF has important clinical significance, and improved awareness of this disease will contribute to prevention of unnecessary thoracotomies.

To explore the potential roles of mitochondrial DNA somatic mutations in benign breast disease based on the entire genome of mitochondrial DNA and elucidate the relationship between benign breast disease and breast cancer.The genomic DNA of tumor tissue and peripheral blood in 28 benign breast disease patients with an average age of 33 years (range: 30 - 50) were extracted respectively. According to the revised Cambridge reference sequence and phylogenetic tree reconstruction, the mutations were identified and distinction was made between somatic mutations and private mitochondrial DNA mutations by haplogroup.Seven somatic mutations were detected. One mutation was located in the control region whereas the other six lied in the coding region. Further analyses revealed that, out of these 6 coding-region mutations, 4 were non-synonymous and would introduce the changes of amino acids.The mutations of mitochondrial DNA may play potential roles in the occurrence and development of benign breast disease.