Establishment and application of a reverse dot blot assay for 13 mutations of hearing-loss genes in primary hospitals in China.

Background: Hearing loss is a common sensorineural dysfunction with a high incidence in China. Although genetic factors are important causes of hearing loss, hearing-related gene detection has not been widely adopted in China. Objective: Establishing a rapid and efficient method to simultaneously detect hotspot hearing loss gene mutations. Methods: A reverse dot blot assay combined with a flow-through hybridization technique was developed for the simultaneous detection of 13 hotspot mutations of 4 hearing loss-related genes including GJB2, GJB3, SLC26A4, and the mitochondrial gene MT-RNR1. This method involved PCR amplification systems and a hybridization platform. Results: The technique can detect 13 hotspot mutations of 4 hearing loss-related genes. And a total of 213 blood samples were used to evaluate the availability of this method. Discussion: Our reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify hotspot mutations of 4 hearing loss-related genes in a Chinese population.

Study on Screening Core Biomarkers of Noise and Drug-Induced Hearing Loss Based on Transcriptomics.

Background Noise and drug-induced hearing loss (HL) is becoming more and more serious, but the integration and analysis based on transcriptomics and proteomics are lacking. On the one hand, this study aims to integrate existing public transcriptomic data on noise and gentamicin-induced HL. On the other hand, the study aims to establish the gentamicin and noise-induced HL model of guinea pigs, then to perform the transcriptomic and proteomic analyses. Through comprehensive analysis of the above data, we aim to screen, predict, and preliminarily verify biomarkers closely related to HL. Material and Methods We screened the Gene Expression Omnibus database to obtain transcriptome data expression profiles of HL caused by noise and gentamicin, then constructed the guinea pig HL model and perform the transcriptomic and proteomic analyses. Differential expression and enrichment analysis were performed on public and self-sequenced data, and common differentially expressed genes (DEGs) and signaling pathways were obtained. Finally, we used proteomic data to screen for common differential proteins and validate common differential expression genes for HL. Results By integrating the public data set with self-constructed model data set, we eventually obtained two core biomarkers of HL, which were RSAD2 and matrix metalloproteinase-3 (MMP3). Their main function is to regulate the development of sense organ in the inner ear and they are mainly involved in mitogen-activated protein kinase and phosphoinositol-3 kinase/protein kinase B signaling pathways. Finally, by integrating the proteomic data of the self-constructed model, we also found differential expression of MMP3 protein. This also preliminarily and partially verified the above-mentioned core biomarkers. Conclusion and Significance In this study, public database and transcriptomic data of self-constructed model were integrated, and we screened out two core genes and various signal pathways of HL through differential analysis, enrichment analysis, and other analysis methods. Then, we preliminarily validated the MMP3 by proteomic analysis of self-constructed model. This study pointed out the direction for further laboratory verification of key biomarkers of HL, which is of great significance for revealing the core pathogenic mechanism of HL.

NT5E upregulation in head and neck squamous cell carcinoma: A novel biomarker on cancer-associated fibroblasts for predicting immunosuppressive tumor microenvironment.

Despite tremendous progress made in the diagnosis and managements, head and neck squamous cell carcinoma (HNSC) remains a global medical dilemma with dismal clinical prognosis and high mortality. Gene NT5E encodes the ecto-5'-nucleotidase (CD73), which facilitates the formation of immunosuppressive tumor microenvironment (TME) permissive for tumor progression in various malignancies. Nevertheless, the cell subsets NT5E expressed on and the potential function of NT5E in the TME of HNSC remain virgin lands in HNSC. In this study, we comprehensively performed integrated prognostic analysis and elucidated that NT5E was an independent prognostic indicator for HNSC, for which a high NT5E level predicted poor overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) in HNSC patients (p<0.05). Enrichment analyses revealed the close correlation between NT5E and ECM remodeling, and the latent function of NT5E may involve in epithelial-to-mesenchymal transition (EMT) and metastasis during HNSC progression. HNSC-related immune infiltration analysis and single-cell type analysis demonstrated that NT5E expression was significantly positively associated with cancer-associated fibroblasts (CAFs) in HNSC (p<0.01). NT5E-related TME analysis revealed that NT5E-high group are characterized by low neoantigen loads (NAL, p<0.001) and tumor mutation burden (TMB, p<0.01), indicating high-NT5E-expression HNSC patients may be recalcitrant to immunotherapy. In-situ multicolor immunofluorescence staining was later conducted and the results further verified our findings. Taken together, NT5E could be a novel biomarker in HNSC. Predominantly expressed on CAFs, the upregulation of NT5E might predict an immunosuppressive TME for HNSC patients who may benefit little from immunotherapy. Targeting CAFs with high NT5E expression might be a novel therapeutic strategy for HNSC patients.

The Role of Genetic Variants in the Susceptibility of Noise-Induced Hearing Loss.

Noised-induced hearing loss (NIHL) is an acquired, progressive neurological damage caused by exposure to intense noise in various environments including industrial, military and entertaining settings. The prevalence of NIHL is much higher than other occupational injuries in industrialized countries. Recent studies have revealed that genetic factors, together with environmental conditions, also contribute to NIHL. A group of genes which are linked to the susceptibility of NIHL had been uncovered, involving the progression of oxidative stress, potassium ion cycling, cilia structure, heat shock protein 70 (HSP70), DNA damage repair, apoptosis, and some other genes. In this review, we briefly summarized the studies primary in population and some animal researches concerning the susceptible genes of NIHL, intending to give insights into the further exploration of NIHL prevention and individual treatment.

[Preparation and effect against cervical cancer invasion in vitro of harmine-loaded photosensitive liposomes].

Despite the development of HPV vaccines and screening programs, cervical cancer is still a serious threat to women's health. Early-stage cervical cancer is mainly treated by surgery. However, considering the serious complications after surgery, hyperthermia is recommended to enhance the effect of chemotherapy, retain the integrity of cervix, improve the treatment effect, which provides a therapeutic basis for the early treatment of cervical cancer. The photosensitive liposomes containing harmine and dye IR-780 were prepared by thin-film dispersion method and separated by Sephadex G-50 dextran gel column. The preparation conditions were optimized as the mass ratio of phospholipid to cholesterol membrane material being 8∶1 and that of drug to lipid being 1∶20. The results of HPLC showed that the encapsulation efficiency of harmine was 55.6%±0.18%. The prepared photosensitive liposomes were round and evenly distributed under transmission electron microscope, with the particle size of(125.2±0.62) nm determined by Marvin particle size analyzer and the Zeta potential of(-2.55±0.76) mV. Additionally, the photosensitive liposomes had the photothermal conversion efficiency, an important property of photothermal agent, of 27.1%±0.86%. The photosensitive liposomes stored at 4 ℃ showed stable encapsulation efficiency in the first 14 days without flocculation. The sulforhodamine B(SRB) assay was employed to determine the inhibitory effect of the liposomes on the proliferation of HeLa cells under near-infrared(NIR) irradiation or not, which showcased stronger inhibitory effect under NIR irradiation. The results of Transwell assay indicated that the prepared liposomes significantly inhibited the invasion and migration of HeLa cells in vitro. The findings of this study provide a basis for the treatment of cervical cancer with harmine.

Involvement of NLRP3-inflammasome pathway in noise-induced hearing loss.

The inflammasome is a multiprotein oligomer in the cell cytoplasm and is part of the innate immune system. It plays a crucial role in the pathological process of noise-induced hearing loss (NIHL). However, the mechanisms of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in NIHL have not been clearly demonstrated. In this study, miniature pigs were exposed to white noise at 120 dB(A) and auditory brainstem response measurements were used to measure their hearing function. Immunofluorescence staining, confocal laser scanning microscopy, western blot assay, and quantitative reverse transcription-polymerase chain reaction were used to analyze inflammasome-related protein distribution and expression. NLRP3, interleukin-1ß, interleukin-18, and cleaved-caspase-1 were highly expressed in the cochlea after 120 dB(A) white noise exposure. Our findings suggest that NLRP3-inflammasomes in the cochlea may be activated after acoustic trauma, which may be an important mechanism of noise-induced hearing loss.

[Effect of Sishen Pills and its split prescriptions on Tfr/Tfh9/Tfh17 cells in colitis mice].

This study aims to investigate the regulatory effect of Sishen Pills(SSP) and its split prescriptions Ershen Pills(EP) and Wuweizi Powder(WP) on T follicular helper(Tfh) cell subset in the dextran sodium sulfate(DSS)-induced colitis mice and the mechanism. A total of 60 male SPF BALB/c mice were used, 10 of which were randomly selected as the normal group. The rest 50 were induced with 3% DSS solution for colitis modeling. After modeling, they were randomized into 5 groups: model group, SSP group, EP group, WP group, and mesalazine group. Body mass, colon mass, colon mass index, colon length, and unit colon mass index in each group were observed. After hematoxylin-eosin(HE) staining, the pathological injury of colon tissue was scored. The expression levels of molecules related to the STAT/SOCS signaling pathway in colon tissues were analyzed by Western blot. Differentiation levels of Tfh cells such as CD4~+CXCR5~+IL-9~+(Tfh9), CD4~+CXCR5~+IL-17~+(Tfh17), and CD4~+CXCR5~+Foxp3~+(Tfr) in peripheral blood of mice were detected by flow cytometry. The results showed each treatment group demonstrated significant increase in body mass and colon length, decrease in colon mass, colon mass index, unit colon mass index, and histopathological score(P<0.05, P<0.01), reduction of the expression of p-STAT3, STAT3, p-STAT6, and STAT6(P<0.05, P<0.01), rise of the expression of SOCS1 and SOCS3(P<0.05, P<0.01), decrease of Tfh9 and Tfh17 cells, and increase of Tfr cells(P<0.05, P<0.01) compared with the model group. These results indicated that SSP and the split EP and WP may alleviate ulcerative colitis by inhibiting the activation of STAT/SOCS signaling pathway and regulating the balance of Tfr/Tfh9/Tfh17 cells.

Sox10 Gene Is Required for the Survival of Saccular and Utricular Hair Cells in a Porcine Model.

Pathological changes of the cochlea and hearing loss have been well addressed in Waardenburg syndrome (WS). However, the vestibular organ malformation in WS is still largely unknown. In this study, the differentiation and development of vestibular sensory epithelium and vestibular function caused by SOX10 mutation, a critical gene induces WS, have been studied in minature pig model. Degeneration of vestibular hair cells was found in this Sox10 mutation porcine model. Inner ear phenotype of the SOX10+/R109W miniature pigs showed cochlear abnormalities as well as saccular hypofunction. In the mutant pigs, no prominent dissimilarity was shown in the bone structure of the semicircular canals. However, the saccular membrane was collapsed, and the infusion of stereocilia of the hair cells was observed. There were no dark cells in the utricles in the mutant pigs. The density of the utricular hair cells was also significantly lower in the mutant pigs compared to the wild type. Our study demonstrated that the SOX10 gene and melanocytes play important roles in the vestibular organ development. Sox10 mutation disrupts the KIT-DCT signaling pathway, affects the development of melanocytes, and leads to vestibule morphogenesis.

Curcumin Inhibits T Follicular Helper Cell Differentiation in Mice with Dextran Sulfate Sodium (DSS)-Induced Colitis.

Follicular helper T cells (Tfh) regulate the differentiation of germinal center B cells and maintain humoral immunity. Notably, imbalances in Tfh differentiation often lead to the development of autoimmune diseases, including inflammatory bowel disease (IBD). Curcumin, a natural product derived from Curcuma longa, is effective in relieving IBD in humans and animals, and its mechanisms of immune regulation need further elaboration. In this study, dextran sodium sulfate induced ulcerative colitis in BALB/c mice, and curcumin was administered simultaneously for 7 days. Curcumin effectively upregulated the change rate of mouse weight, colonic length, down-regulated colonic weight, index of colonic weight, colonic damage score and the levels of pro-inflammatory cytokines IL-6, IL-12, IL-23 and TGF-[Formula: see text]1 in colonic tissues of colitis mice. Importantly, curcumin regulated the differentiation balance of Tfh and their subpopulation in colitis mice; the percentages of Tfh (CD4[Formula: see text]CXCR5[Formula: see text]BCL-6[Formula: see text], CD4[Formula: see text]CXCR5[Formula: see text]PD-1[Formula: see text], CD4[Formula: see text]CXCR5[Formula: see text]PD-L1[Formula: see text], CD4[Formula: see text]CXCR5[Formula: see text]ICOS[Formula: see text], Tfh17 and Tem-Tfh were downregulated significantly, while CD4[Formula: see text]CXCR5[Formula: see text]Blimp-1[Formula: see text], Tfh1, Tfh10, Tfh21, Tfr, Tcm-Tfh and Tem-GC Tfh were upregulated. In addition, curcumin inhibited the expression of Tfh-related transcription factors BCL-6, p-STAT3, Foxp1, Roquin-1, Roquin-2 and SAP, and significantly upregulated the protein levels of Blimp-1 and STAT3 in colon tissue. In conclusion, curcumin may be effective in alleviating dextran sulfate sodium-induced colitis by regulating Tfh differentiation.

Involvement of Cholesterol Metabolic Pathways in Recovery from Noise-Induced Hearing Loss.

The objective of this study was to explore the molecular mechanisms of acute noise-induced hearing loss and recovery of steady-state noise-induced hearing loss using miniature pigs. We used miniature pigs exposed to white noise at 120 dB (A) as a model. Auditory brainstem response (ABR) measurements were made before noise exposure, 1 day and 7 days after noise exposure. Proteomic Isobaric Tags for Relative and Absolute Quantification (iTRAQ) was used to observe changes in proteins of the miniature pig inner ear following noise exposure. Western blot and immunofluorescence were performed for further quantitative and qualitative analysis of proteomic changes. The average ABR-click threshold of miniature pigs before noise exposure, 1 day and 7 days after noise exposure, were 39.4 dB SPL, 67.1 dB SPL, and 50.8 dB SPL, respectively. In total, 2,158 proteins were identified using iTRAQ. Both gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses showed that immune and metabolic pathways were prominently involved during the impairment stage of acute hearing loss. During the recovery stage of acute hearing loss, most differentially expressed proteins were related to cholesterol metabolism. Western blot and immunofluorescence showed accumulation of reactive oxygen species and nuclear translocation of NF-κB (p65) in the hair cells of miniature pig inner ears during the acute hearing loss stage after noise exposure. Nuclear translocation of NF-κB (p65) may be associated with overexpression of downstream inflammatory factors. Apolipoprotein (Apo) A1 and Apo E were significantly upregulated during the recovery stage of hearing loss and may be related to activation of cholesterol metabolic pathways. This is the first study to use proteomics analysis to analyze the molecular mechanisms of acute noise-induced hearing loss and its recovery in a large animal model (miniature pigs). Our results showed that activation of metabolic, inflammatory, and innate immunity pathways may be involved in acute noise-induced hearing loss, while cholesterol metabolic pathways may play an important role in recovery of hearing ability following noise-induced hearing loss.

The value of multidisciplinary team in syncope clinic for the effective diagnosis of complex syncope.

BACKGROUND: Syncope is a perplexing challenge that often receives thorough evaluation, yet the diagnosis remains unclear. Usually, the emergency department is the first point at which patients present with syncope. However, diverse medical factors, including low diagnostic rates and inconsistent management by doctors, add to healthcare costs and delay diagnosis for syncope patients. METHODS: Patients who had been to the emergency department at least once but were not given a clear diagnosis of syncope were recruited into our study at the time they visited syncope clinic staffed by a multidisciplinary team. Complete medical histories and clinical examinations were conducted by both experienced cardiologists and neurologists. If patients were not given a conclusive diagnosis at the syncope clinic on the basis of outpatient examinations, they were admitted for further evaluation. RESULTS: A total of 209 consecutive patients claiming "syncope" visited the syncope clinic, yet only 167 patients were formally diagnosed with syncope. For these 167 patients, the mean age was 55.93 ± 17.40 years old, and 41.3% were male. The proportions of cardiac syncope, reflex syncope, orthostatic hypotension (OH), and syncope of uncertain etiology were 19.8%, 64.1%, 7.8%, and 8.4%, respectively. The diagnostic rate was 91.6%, and the hospitalization rate was 23.4%. Patients with reflex syncope and OH were younger than patients with cardiac syncope. Cardiac syncope tends to occur more frequently in males, while reflex syncope is more likely in females. CONCLUSIONS: The cooperation of professional cardiologists and neurologists will play an important role in improving diagnostic rates, lowering admission rates, and reducing medical costs.

Establishment of a Large Animal Model for Eustachian Tube Functional Study in Miniature Pigs.

This study was performed to investigate whether miniature pigs are a suitable animal model for studies of the Eustachian tube (ET). Sixteen Chinese experimental miniature pigs were used in this investigation. Ten animals were used for anatomical and morphometric analyses to obtain qualitative and quantitative information regarding the ET. Three animals were used for histological analysis to determine the fine structure of ET cross-sections. Three animals were used to investigate the feasibility of balloon dilation of the Eustachian tube (BDET). The anatomical study indicated that the pharyngeal orifice and tympanic orifice of the miniature pig ET are located at the posterior end of the nasal lateral wall and anterior wall of the middle ear cavity, respectively. The cartilaginous tube was seen to pass through the whole length of the ET, the length of the cartilaginous part of the ET and the diameter of the isthmus were similar between humans and miniature pigs. The inclination of the ET in miniature pigs was larger than that in humans. The gross histology seemed to be slightly different between miniature pig and human, but the fine structures were essentially the same in both species. BDET experiments verified that the miniature pig model is suitable as a model for clinical operations. The miniature pig ET corresponds very well to that of humans. In addition, the miniature pig ET is suitable as a model for clinical operations. Therefore, the miniature pig is a valid animal model for ET study. Anat Rec, 302:1024-1038, 2019. © 2019 Wiley Periodicals, Inc.

Degeneration of saccular hair cells caused by MITF gene mutation.

BACKGROUND: Waardenburg syndrome (WS) is the consequence of an inherited autosomal dominant mutation which causes the early degeneration of intermediate cells of cochlear stria vascularis (SV) and profound hearing loss. Patients with WS may also experience primary vestibular symptoms. Most of the current WS studies did not discuss the relationship between WS and abnormal vestibular function. Our study found that a spontaneous mutant pig showed profound hearing loss and depigmentation. MITF-M, a common gene mutation causes type WS which affect the development of the intermediate cell of SV, was then identified for animal modeling. RESULTS: In this study, the degeneration of vestibular hair cells was found in pigs with MITF-M. The morphology of hair cells in vestibular organs of pigs was examined using electron microscopy from embryonic day E70 to postnatal two weeks. Significant hair cell loss in the mutant saccule was found in this study through E95 to P14. Conversely, there was no hair cell loss in either utricle or semi-circular canals. CONCLUSIONS: Our study suggested that MITF-M gene mutation only affects hair cells of the saccule, but has no effect on other vestibular organs. The study also indicated that the survival of cochlear and saccular hair cells was dependent on the potassium release from the cochlear SV, but hair cells of the utricle and semi-circular canals were independent on SV.

Key Genes and Pathways Associated With Inner Ear Malformation in SOX10  p.R109W Mutation Pigs.

SRY-box 10 (SOX10) mutation may lead to inner ear deformities. However, its molecular mechanisms on inner ear development are not clear. In this work, the inner ear morphology was investigated at different embryonic stages of the SOX10 mutation miniature porcine model with sensorineural hearing loss, and high-throughput RNA-seq and bioinformatics analyses were applied. Our results indicated that the SOX10 mutation in the miniature pigs led to an incomplete partition (IP) of the cochlea, a cystic apex caused by fusion from middle and apical turns, cochlear modiolar defects and a shortened cochlear duct. The model demonstrated 173 differentially expressed genes (DEGs) and 185 differentially expressed long non-coding RNAs (lncRNAs). The down-regulated DEGs most significantly enriched the inflammatory mediator regulation of the TRP channels, arachidonic acid metabolism, and the salivary secretion pathways, while the up-regulated DEGs most significantly enriched the systemic lupus erythematosus and alcoholism pathways. Based on gene cluster analysis, we selected four gene groups: WNT1, KCNQ4, STRC and PAX6.

Modulation of Glucose Takeup by Glucose Transport on the Isolated OHCs.

Glucose is a fundamental source of energy for mammalian cells; however, whether glucose is taken up through the lateral walls of cochlear outer hair cells (OHCs) is unknown. The OHC lateral wall is complex, composed of a plasma membrane, cortical lattice, and subsurface cisternae. This study assessed the uptake of glucose by OHCs using live-cell microscopy and examined the distribution of glucose transporter isoforms by immunohistochemistry. We found that glucose transporter-4 was mostly expressed on the lateral wall of OHCs. Glucose crossed the lateral walls of OHCs via glucose transporters-4 mainly, and this process could be modulated. These results suggest that the lateral walls are involved in modulating energy transport into OHCs.

Inner ear structure of miniature pigs measured by multi-planar reconstruction techniques.

To study the structures of the scala vestibuli and tympani of miniature pigs in order to evaluate the feasibility of using miniature pigs as the animal model for cochlear implant. The temporal bones of three miniature pigs with normal hearing were scanned by micro-CT. With the aid of the Mimics software, we reconstructed the 3D structure of inner ear basing on the serial images of the miniature pig, and obtained dimensions of the scala vestibuli and tympani with multi-planar reconstruction (MPR) technique. The constructed slicing images displayed the fine structures of the cochlea. The results of our study showed that the cross-sectional areas of the scala tympani were greatest at 2.67 ± 0.90 mm2 when the circumferential length from the starting point of basal turn of the cochlea reached to 1.16 mm. The scala vestibuli has a largest width and height at the starting point of basal turn. The width and the height were 2.65 ± 0.45 mm and 2.43 ± 0.2 mm respectively. The largest width and height of the scala tympani were 2.17 ± 0.30 mm and 1.83 ± 0.42 mm. The result of our study suggests that the cochlea of miniature pigs is highly consistent with human's. Miniature pigs may be used as a new model for cochlear implant. MPR technique may be used as a new approach to obtain further information of patient's cochlea in surgeons which is helpful to select suitable cochlear implant devices and surgery approach.

A hypothesis study on bionic active noise reduction of auditory organs.

BACKGROUND: Noise exposure can lead to hearing loss and multiple system dysfunctions. As various forms of noise exist in our living environments, and our auditory organs are very sensitive to acoustic stimuli, it is a challenge to protect our hearing system in certain noisy environments. PRESENTATION OF THE HYPOTHESIS: Herein, we propose that our hearing organ could serve as a noise eliminator for high intensity noise and enhance acoustic signal processing abilities by increasing the signal-noise ratio. For suprathreshold signals, the hearing system is capable of regulating the middle ear muscles and other structures to actively suppress the sound level to a safe range. TESTING THE HYPOTHESIS: To test our hypothesis, both mathematic model analyses and animal model studies are needed. Based on a digital 3D reconstructed model, every structure in the auditory system can be analyzed and tested for its contribution to the process of noise reduction. Products manufactured by this bionic method could be used and verified in animal models and volunteers. IMPLICATIONS: By mimicking the noise-reduction effect of the sophisticated structures in the hearing system, we may be able to provide a model that establishes a new active-sound-suppression mode. This innovative method may overcome the limited capabilities of current noise protection options and become a promising possibility for noise prevention.

Analysis of EGFR, EML4-ALK, KRAS, and c-MET mutations in Chinese lung adenocarcinoma patients.

INTRODUCTION: Mutation analysis of cancer driver genes is helpful for determining an optimal treatment strategy. We evaluated mutations in four driver genes, namely epidermal growth factor receptor (EGFR), Kirsten ras oncogene (KRAS), c-MET, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), in Chinese lung adenocarcinoma patients from Hunan Province. METHODS: We enrolled 110 lung adenocarcinoma patients in a single institution. EGFR and KRAS mutations were examined by direct sequencing, the EML4-ALK fusion gene was analyzed by fluorescence in situ hybridization, and c-MET amplification and c-Met protein expression were detected by quantitative PCR and immunohistochemistry, respectively. RESULTS: EGFR and KRAS mutations were observed in 52.7% (58/110) and 3.6% (4/106) of patients, respectively. c-MET amplification was detected in 5.5% (6/110) of patients. In addition, 30% (33/110) of the cases expressed c-Met protein, including all of the patients harboring c-MET amplification. Ten percent (11/110) of patients harbored the EML4-ALK fusion gene, and the frequency of ALK rearrangement was higher than that of other cohort analyses involving patients from other regions in China. Almost all of these gene mutations were exclusive except that in two female non-smoking patients, who harbored an EGFR mutation and EML4-ALK rearrangement simultaneously. In total, 70% of patients in the study harbored one of the four gene mutations. CONCLUSIONS: Most Chinese lung adenocarcinoma patients harbor driver gene mutations, among which ALK rearrangements were more common in Hunan patients than in previously reported populations. Future clinical trials should be conducted to determine the safety and efficacy of drug combination targeting different driver mutations.