Solobacterium moorei promotes the progression of adenomatous polyps by causing inflammation and disrupting the intestinal barrier.

BACKGROUND: Adenomatous polyps (APs) with inflammation are risk factors for colorectal cancer. However, the role of inflammation-related gut microbiota in promoting the progression of APs is unknown. METHODS: Sequencing of the 16S rRNA gene was conducted to identify characteristic bacteria in AP tissues and normal mucosa. Then, the roles of inflammation-related bacteria were clarified by Spearman correlation analysis. Furthermore, colorectal HT-29 cells, normal colon NCM460 cells, and azoxymethane-treated mice were used to investigate the effects of the characteristic bacteria on progression of APs. RESULTS: The expression levels of inflammation-related markers (diamine oxidase, D-lactate, C-reactive protein, tumor necrosis factor-α, interleukin-6 and interleukin-1ß) were increased, whereas the expression levels of anti-inflammatory factors (interleukin-4 and interleukin-10) were significantly decreased in AP patients as compared to healthy controls. Solobacterium moorei (S. moorei) was enriched in AP tissues and fecal samples, and significantly positively correlated with serum inflammation-related markers. In vitro, S. moorei preferentially attached to HT-29 cells and stimulated cell proliferation and production of pro-inflammatory factors. In vivo, the incidence of intestinal dysplasia was significantly increased in the S. moorei group. Gavage of mice with S. moorei upregulated production of pro-inflammatory factors, suppressed proliferation of CD4+ and CD8+cells, and disrupted the integrity of the intestinal barrier, thereby accelerating progression of APs. CONCLUSIONS: S. moorei accelerated the progression of AP in mice via activation of the NF-κB signaling pathway, chronic low-grade inflammation, and intestinal barrier disruption. Targeted reduction of S. moorei presents a potential strategy to prevent the progression of APs.

mRNA-Laden Lipid-Nanoparticle-Enabled in Situ CAR-Macrophage Engineering for the Eradication of Multidrug-Resistant Bacteria in a Sepsis Mouse Model.

Sepsis, which is the most severe clinical manifestation of acute infection and has a mortality rate higher than that of cancer, represents a significant global public health burden. Persistent methicillin-resistant Staphylococcus aureus (MRSA) infection and further host immune paralysis are the leading causes of sepsis-associated death, but limited clinical interventions that target sepsis have failed to effectively restore immune homeostasis to enable complete eradication of MRSA. To restimulate anti-MRSA innate immunity, we developed CRV peptide-modified lipid nanoparticles (CRV/LNP-RNAs) for transient in situ programming of macrophages (MΦs). The CRV/LNP-RNAs enabled the delivery of MRSA-targeted chimeric antigen receptor (CAR) mRNA (SasA-CAR mRNA) and CASP11 (a key MRSA intracellular evasion target) siRNA to MΦs in situ, yielding CAR-MΦs with boosted bactericidal potency. Specifically, our results demonstrated that the engineered MΦs could efficiently phagocytose and digest MRSA intracellularly, preventing immune evasion by the "superbug" MRSA. Our findings highlight the potential of nanoparticle-enabled in vivo generation of CAR-MΦs as a therapeutic platform for multidrug-resistant (MDR) bacterial infections and should be confirmed in clinical trials.

Pyrrole-Doped Polydopamine-Pyrrole (PDA-nPY) Nanoparticles with Tunable Size and Improved NIR Absorption for Photothermal Therapy.

Polydopamine (PDA) as a melanin-like biomimetic material with excellent biocompatibility, full spectrum light absorption capacity and antioxidation property has been extensively applied in the biomedical field. Based on the high reactivity of dopamine (DA), exploiting new strategies to fabricate novel PDA-based nano-biomaterials with controllable size and improved performance is valuable and desirable. Herein, we reported a facile way to synthesize pyrrole-doped polydopamine-pyrrole nanoparticles (PDA-nPY NPs) with tunable size and enhanced near-infrared (NIR) absorption capacity through self-oxidative polymerization of DA with PY in an alkaline ethanol/H2O/NH4OH solution. The PDA-nPY NPs maintain excellent biocompatibility and surface reactivity as PDA. By regulating the volume of added PY, PDA-150PY NPs with a smaller size (<100 nm) and four-fold higher absorption intensity at 808 nm than that of PDA can be successfully fabricated. In vitro and in vivo experiments effectively further demonstrate that PDA-150PY NPs can effectively inhibit tumor growth and completely thermally ablate a tumor. It is believed that these PY doped PDA-nPY NPs can be a potential photothermal (PT) agent in biomedical application.

Clinically Translatable Solid-State Dye for NIR-II Imaging of Medical Devices.

Medical devices are commonly implanted underneath the skin, but how to real-time noninvasively monitor their migration, integrity, and biodegradation in human body is still a formidable challenge. Here, the study demonstrates that benzyl violet 4B (BV-4B), a main component in the FDA-approved surgical suture, is found to produce fluorescence signal in the first near-infrared window (NIR-I, 700-900 nm) in polar solutions, whereas BV-4B self-assembles into highly crystalline aggregates upon a formation of ultrasmall nanodots and can emit strong fluorescence in the second near-infrared window (NIR-II, 1000-1700 nm) with a dramatic bathochromic shift in the absorption spectrum of ≈200 nm. Intriguingly, BV-4B-involved suture knots underneath the skin can be facilely monitored during the whole degradation process in vivo, and the rupture of the customized BV-4B-coated silicone catheter is noninvasively diagnosed by NIR-II imaging. Furthermore, BV-4B suspended in embolization glue achieves hybrid fluorescence-guided surgery (hybrid FGS) for arteriovenous malformation. As a proof-of-concept study, the solid-state BV-4B is successfully used for NIR-II imaging of surgical sutures in operations of patients. Overall, as a clinically translatable solid-state dye, BV-4B can be applied for in vivo monitoring the fate of medical devices by NIR-II imaging.

Surficial nano-deposition locoregionally yielding bactericidal super CAR-macrophages expedites periprosthetic osseointegration.

Tracking and eradicating Staphylococcus aureus in the periprosthetic microenvironment are critical for preventing periprosthetic joint infection (PJI), yet effective strategies remain elusive. Here, we report an implant nanoparticle coating that locoregionally yields bactericidal super chimeric antigen receptor macrophages (CAR-MΦs) to prevent PJI. We demonstrate that the plasmid-laden nanoparticle from the coating can introduce S. aureus-targeted CAR genes and caspase-11 short hairpin RNA (CASP11 shRNA) into macrophage nuclei to generate super CAR-MΦs in mouse models. CASP11 shRNA allowed mitochondria to be recruited around phagosomes containing phagocytosed bacteria to deliver mitochondria-generated bactericidal reactive oxygen species. These super CAR-MΦs targeted and eradicated S. aureus and conferred robust bactericidal immunologic activity at the bone-implant interface. Furthermore, the coating biodegradability precisely matched the bone regeneration process, achieving satisfactory osteogenesis. Overall, our work establishes a locoregional treatment strategy for priming macrophage-specific bactericidal immunity with broad application in patients suffering from multidrug-resistant bacterial infection.

Enantioselective C(sp3)-H Functionalization of Oxacycles via Photo-HAT/Nickel Dual Catalysis.

The selective functionalization of ubiquitous but inert C-H bonds is highly appealing in synthetic chemistry, but the direct transformation of hydrocarbons lacking directing groups into high-value chiral molecules remains a formidable challenge. Herein, we develop an enantioselective C(sp3)-H functionalization of undirected oxacycles via photo-HAT/nickel dual catalysis. This protocol provides a practical platform for the rapid construction of high-value and enantiomerically enriched oxacycles directly from simple and abundant hydrocarbon feedstocks. The synthetic utility of this strategy is further demonstrated in the late-stage functionalization of natural products and the synthesis of many pharmaceutically relevant molecules. Experimental and density functional theory calculation studies provide detailed insights into the mechanism and the origin of enantioselectivity for the asymmetric C(sp3)-H functionalization.

Proteomic and Phosphoproteomic Analyses Reveal the Oncogenic Role of PTK7-NDRG1 Axis in Non-small-cell Lung Cancer Cell Resistance to AZD9291.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important chemotherapeutics for non-small-cell lung cancer (NSCLC) therapy. The resistance to EGFR-TKIs is one of the biggest obstacles to NSCLC outcome. In this study, taking advantage of phospho- and proximal proteomic techniques, we analyzed the network rearrangement in cell lines responding to AZD9291 treatment and found that cell-cell adhesion was dramatically enhanced in AZD9291-resistant cells. Further analysis revealed that protein tyrosine kinase 7 (PTK7) expression was significantly elevated. Knockdown or overexpression assays showed that PTK7 played a critical role in improving cell adhesion, which enhanced drug resistance. Because PTK7 is a membrane-localized pseudokinase, the proximal labeling probe BirA* was fused to reveal PTK7-interacting proteins. We found that PTK7 interacted with and stabilized NDRG1, which is located predominantly adjacent to adherens junctions. Downregulation of PTK7 or NDRG1 eliminated the resistance of H1975-resistant (H1975-R) and PC9-resistant (PC9-R) cells to AZD9291, suggesting that the PTK7-NDRG1 axis might be a potential target to eliminate the EGFR-TKI resistance during NSCLC therapy.

Study on the Microstructure and Mechanical Properties of Diamond Particle-Reinforced Copper-Iron Sandwich Composites Prepared by Powder Metallurgy.

Synthetic diamond particle-reinforced copper-iron composites (SD/Cu-Fe) were produced by the powder metallurgical method for stone cutting applications, and the microstructure, density, compactness, hardness, flexure strength, and wear resistance of the composites were characterized in this work. The results showed that the diamond particles were relatively uniformly distributed in most areas of the copper matrix and the crystal shape of diamond particles were relatively intact in the sintering temperature range from 740 °C to 780 °C. The interfaces between the diamond particles and copper matrix, as well as the interfaces between the copper matrix and iron layer, were well bonded without significant gaps. The physical properties of composites increased first and then decreased with the sintering temperature. When the sintering temperature was 770 °C, the related properties reached the best. Diamond played a key role in improving the properties of the SD/Cu-Fe sandwich composite. This work provides a basis for the research and development of high-performance diamond-reinforced copper-based iron sandwich composites.

Comparison of cervical neck strain in common otolaryngology surgeries.

BACKGROUND: Cervical neck strain and surgical ergonomics is an increasingly important topic being addressed in this time and age. With new technologies, visualizations, and approaches to surgeries, there are now different strains and duration of strains to the cervical neck. Recently the effect of chronic cell phone use has been described as "text neck." In a similar fashion we understand that certain otolaryngology surgeries can also impart chronic strain to the cervical neck. We aim to quantitatively describe strain for different types of surgeries by looking at posture, duration of surgery, and anatomic ergonomics of specific surgeries. METHODS: Lateral photo documentation of posture during 6 common otolaryngology procedures, used to estimate cervical neck angle and calculate force and impulse to cervical neck. RESULTS: Six common otolaryngology procedures show various cervical neck angles ranging from around 0° to 60° of neck flexion, with subsequent forces ranging from 16 lb to 60 lb of force. When accounting for surgical time, bigger differences arose with impulses ranging from 270,000 N∗s to 3,300,000 N∗s. Noticeably, thyroidectomy and cleft palate showed much higher impulses than the other four types of surgeries. CONCLUSION: Both cervical neck flexion and duration of surgery play important roles in total neck theoretical strain. Variance exists between neck strains of common otolaryngology surgeries. There is a necessity for continued study and improvement in surgical ergonomics.

Catalpol protects AC16 cells from hypoxia/reoxygenation injury by regulating the miR-22-3p/DPP4 axis.

Catalpol (CA) is widely used in the protection of cardiomyocytes. Nevertheless, the mechanism of CA in alleviating ischemia-reperfusion-induced injury of cardiomyocytes remains unclear. Human cardiomyocyte AC16 cells were subjected to hypoxia/reoxygenation (H/R) injury. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were applied to detect tumor necrosis factor-alpha (TNF-α) mRNA, interleukin-6 (IL-6) mRNA, interleukin-1beta (IL-1ß) mRNA, microRNA-22-3p (miR-22-3p), dipeptidyl peptidase 4 (DPP4) mRNA, and DPP4 protein expressions. The cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Lactate dehydrogenase (LDH) and creatine kinase (CK-MB) were examined by enzyme-linked immunosorbent assay (ELISA) kits. A dual-luciferase reporter gene assay was performed to confirm the binding sequence between miR-22-3p and DPP4 mRNA 3'-untranslated region (3'UTR). CA promoted the viability and reduced cell apoptosis of AC16 cells and repressed the release of inflammatory cytokines TNF-α, IL-6, and IL-1ß, and inhibited the leakage of myocardial injury markers LDH and CK-MB. Furthermore, CA enhanced the expression of miR-22-3p in cardiomyocytes, and DPP4 was validated to be the target gene of miR-22-3p. The inhibition of miR-22-3p and augmentation of DPP4 reversed the above effects of CA. CA protects A16 cells from H/R injury by regulating the miR-22-3p/DPP4 axis.

Degradation of nuclear components via different autophagy pathways.

Eukaryotic cells have evolved different modes of autophagy, including macroautophagy and microautophagy, to deliver their own components to lysosomes or vacuoles for degradation. While an increasing body of research has established that autophagy plays pivotal roles for the maintenance and regulation of various cellular constituents, recent studies have begun to reveal that parts of the nucleus, for example, nucleus-derived vesicles and nuclear proteins, also become targets of autophagic degradation in different physiological or pathological contexts, including nutrient deprivation, defective nuclear pore complex (NPC) assembly, DNA damage, cellular senescence, and oncogenic insults. Here, we overview our current knowledge on the mechanisms and physiological roles of these 'nucleophagy' pathways and discuss their possible interplays and remaining issues.

Myosin 1b promotes migration, invasion and glycolysis in cervical cancer via ERK/HIF-1α pathway.

BACKGROUND: Increasing evidence indicated that metabolic reprograming is essential and has been regarded as a hallmark of cancer. Although the biological functions of Myosin 1b (Myo1b) have been reported in several malignancies, the correlation between Myo1b and cancer metabolism, and its underlying mechanisms remain elusive, particularly in cervical cancer (CC). METHODS: Myo1b and other glycolytic enzymes expression levels were examined in CC cells and tumor tissues from xenograft models by quantitative real-time PCR, Western blot and immunohistochemistry. The biological impacts and regulatory mechanisms of Myo1b on cell migration, invasion and glycolysis were explored. Also, the effects of Myo1b on carcinogenesis and metastasis in nude mice were investigated. RESULTS: Upregulation of Myo1b was found in CC tissues and associated with poor prognosis. Overexpressed Myo1b not only significantly elevated CC cell glycolysis, migration and invasion in vitro, but also promoted tumorigenesis and metastasis in vivo. Conversely, Myo1b knockdown had opposite consequences. Moreover, our study suggested that Myo1b stimulated ERK/HIF-1α pathway and its downstream glycolysis associated genes to modulate the glycolysis, migration and invasion of CC. CONCLUSION: These findings provide evidence that Myo1b regulates migration, invasion and glycolysis in CC through ERK/HIF-1α pathway, suggesting a promising remedial target in treatment of CC.

Ocular abnormalities in a large patient cohort with retinitis pigmentosa in Western China.

BACKGROUND: To report the prevalence of ocular abnormalities and investigate visual acuity in a large cohort of retinitis pigmentosa (RP) patients in Western China. METHODS: The medical records and ophthalmic examination reports of 2127 eyes of 1065 RP patients at one eye hospital were retrospectively reviewed to determined the prevalence of ocular abnormalities and the relationship between best corrected visual acuity (BCVA) and macular abnormalities. RESULTS: Nyctalopia (58.2%) and blurred vision (27.1%) were the leading reasons for RP patients to request an ophthalmic examination. BCVA measurements in the better eyes at first clinical presentation showed that 304 patients (28.5%) were categorised as blind and 220 patients (20.7%) as low vision. The most common ocular abnormalities were macular abnormalities (59.7%) and cataracts (43.1%). The macular abnormalities included epiretinal membranes (51.1%), cystoid macular edema (18.4%), vitreomacular traction syndrome (2.4%), macular holes (2.3%) and choroidal neovascular membranes (0.05%). Glaucoma was found in 35 eyes (1.6%). The proportions of epiretinal membranes (p = 0.001) and macular holes (p = 0.008) increased significantly with age. Cystoid macular edema was significantly associated with poorer visual acuity in RP patients with clear lens (p = 0.002). CONCLUSION: Cataracts and macular abnormalities are common in RP patients. Of the macular abnormalities, cystoid macular edema may have a negative effect on BCVA in RP patients with clear lens. Therefore, optical coherence tomography screening in RP patients is highly recommended for early detection and treatment of maculopathy.

Boron-doped diamond (BDD) electro-oxidation coupled with nanofiltration for secondary wastewater treatment: Antibiotics degradation and biofouling.

In this study, a boron-doped diamond (BDD) electro-oxidation technology coupled with nanofiltration membrane (EO-NF) technology was investigated for its effectiveness in removing antibiotics (i.e., sulfamethazine:SMZ) and mitigating biofouling during secondary wastewater treatment. The result showed that EO obtained an effective SMZ removal, owing to the ·OH generation observed by Electron paramagnetic resonance (EPR) analysis; complete elimination of SMZ was found under the high current density (30 mA/cm2) and long Electrolysis Time (ET = 60 min). Meanwhile, EO-NF process enabled to reduce COD content from 60 mg/L to nearly 5 mg/L. Furthermore, regardless of the effect of EO process, NF could retain most NH3-N because of the excellent performance of NF for ions rejection, and its permeate concentration was below 0.5 mg/L. EO was able to reduce membrane fouling notably, increasing the final flux (15 L/(m2·h)) of NF by 25.1% during long-term operation (240 h). Scanning electron microscopy-Energy dispersive spectrometry (SEM-EDS) showed that a porous layer formed on the vicinity of NF membrane in the case of filtrating EO effluent, in contrast to a uniform and dense biofouling layer generated during the direct NF. Besides, the content of adenosine triphosphate (ATP) and the number of bacterial colonies in the retentate of the EO-NF process were greater than those of the direct NF process. This resulted in a smaller amount of extracellular polymeric substances (EPS) attaching to the membrane surface, decreasing the tightness and hardness of the fouling layer in the case of EO, as indicated by CLSM analysis. Overall, considering its ability to effectively eliminate persistent contaminants and reduce membrane fouling, BDD-based EO is considered a promising pre-treatment option for future NF applications.

VarBen: Generating in Silico Reference Data Sets for Clinical Next-Generation Sequencing Bioinformatics Pipeline Evaluation.

Next-generation sequencing is increasingly being adopted as a valuable method for the detection of somatic variants in clinical oncology. However, it is still challenging to reach a satisfactory level of robustness and standardization in clinical practice when using the currently available bioinformatics pipelines to detect variants from raw sequencing data. Moreover, appropriate reference data sets are lacking for clinical bioinformatics pipeline development, validation, and proficiency testing. Herein, we developed the Variant Benchmark tool (VarBen), an open-source software for variant simulation to generate customized reference data sets by directly editing the original sequencing reads. VarBen can introduce a variety of variants, including single-nucleotide variants, small insertions and deletions, and large structural variants, into targeted, exome, or whole-genome sequencing data, and can handle sequencing data from both the Illumina and Ion Torrent sequencing platforms. To demonstrate the feasibility and robustness of VarBen, we performed variant simulation on different sequencing data sets and compared the simulated variants with real-world data. The validation study showed that the simulated data are highly comparable to real-world data and that VarBen is a reliable tool for variant simulation. In addition, our collaborative study of somatic variant calling in 20 laboratories emphasizes the need for laboratories to evaluate their bioinformatics pipelines with customized reference data sets. VarBen may help users develop and validate their bioinformatics pipelines using locally generated sequencing data.

Assessment of significant procedures in multigene molecular detection for breast cancer in clinical laboratories: from variant detection to targeted therapy.

BACKGROUND: In recent years, numerous novel targeted drugs against breast cancer have been developed because of the rapid progress in multigene molecular testing based on next-generation sequencing (NGS). However, it is a great challenge for clinicians to update the drug information timely, therefore necessitating that clinical laboratories provide adequate and comprehensive targeted drugs information to clinicians as a reference. The premise of providing this information is the accuracy of variant detection. Our study aimed to assess the entire process of variant detection, interpretation, and targeted therapy. METHODS: Laboratories were instructed to use routine methods for variant detection. The results were evaluated based on a predefined score system, and differences in variant interpretation were analyzed. Targeted drug information provided by laboratories was also summarized, and its accuracy and sufficiency were assessed. RESULTS: Overall, 90.1% (82/91) of the laboratories produced accurate results. 78.9% (15/19) of the errors were false positives or false negatives. Incorrect and insufficient drug information was mainly provided due to failure in timely database updating, inconsistencies with the detected mutations or given clinical information, and negligence during phase I clinical trials. To prioritize providing targeted drug information, laboratories collected data were based on different factors, including variant clinical significance, allele frequency, and variant positions in the signal pathway. CONCLUSION: The variant detection capability was satisfactory, but the ability to provide accuracy and comprehensive targeted drug information should be urgently improved. Our study summarized a completed NGS-based multigene molecular detection pipeline, aiming to better inform precision treatment for breast cancer patients.

IDO inhibitor synergized with radiotherapy to delay tumor growth by reversing T cell exhaustion.

Previous studies suggest that radiotherapy (RT) can induce immune activation, which not only reduces the progression of tumors, but also causes the release of tumor antigens. The combination of RT and immune checkpoint blockade, such as the inhibition of programmed cell death 1 (PD­1) and programmed cell death ligand 1 (PD­L1), has been demonstrated to yield impressive response rates. However, a substantial proportion of patients develop resistance such therapies. Previous studies have shown that indoleamine 2,3­dioxygenase (IDO) causes T cell exhaustion and increased formation of regulatory T cells (Tregs), upregulating the expression of inhibitory receptors and ligands. Therefore, the application of IDO inhibitors combined with RT may have a synergistic effect by relieving immunosuppression. Lewis lung cancer cell­bearing mice were treated with the IDO inhibitor 1­methyl­tryptophan (1MT) and/or 10 Gy RT. Tumor size was measured every day, flow cytometry was performed to measure the expression of dendritic cell (DC) maturation markers, inhibitory receptors, ligands, cytotoxic T cells and Treg phenotypic markers. Reverse transcription­quantitative PCR was used to evaluate the mRNA expression levels of IDO, PD­L1, PD­1, T cell immunoglobulin domain and mucin domain 3 (TIM­3), B­ and T­lymphocyte attenuator (BTLA) and galectin­9. Compared with the control group, treatment with 1MT or RT reduced tumor growth, however, the combination therapy was more effective than either treatment alone. Flow cytometry showed the upregulation of CD80, CD86 and the major histocompatibility complex II in spleen DCs and the concurrent downregulation of CD4, CD25 and forkhead box protein P3 in lymphocytes in the treatment groups. Compared with the control group, inhibitory receptors and ligands that affect DCs and T cells were observed, expression levels of PD­L1, PD­1, TIM­3, BTLA and galectin­9 are decreased in treatment group compared with control. IDO inhibition synergized with RT to downregulate Tregs, inhibitory receptors and ligands to prevent T cell exhaustion. By activating DCs and T cells, this combination therapy may strongly enhance antitumor immunity and inhibit tumor progression.

Challenges of Providing Concordant Interpretation of Somatic Variants in Non-Small Cell Lung Cancer: A Multicenter Study.

Background: Success of multiple-gene mutation tests by next-generation sequencing (NGS), associated with molecular targeting therapies for cancers, depending on the accuracy and consistency of interpreting variants. Here, we summarized reports from clinical laboratories for cases with non-small cell lung cancer (NSCLC) and discussed conflicting interpretations of somatic variants. Methods: Three mimetic DNA samples, containing six somatic mutations, were prepared based on three clinical case reports of NSCLC. Clinical reports and genetic testing questionnaires were collected from 67 laboratories enrolled in this investigation. Results: Thirty-four laboratories with correct variant results identified two variants, based on FDA approval of targeted drugs for the same tumor, consistently, with strong clinical significance, whereas the other variants were classified with conflicting interpretations. Discordant interpretations were reported for ERBB2 with three different classifications, including strong clinical significance (53.0%, 18/34), potential clinical significance (38.2%, 13/34), and unknown significance (8.8%, 3/34). In the variant therapeutic drug recommendation section, 32.4% of the laboratories (11/34) did not recommend all the available therapeutic drugs designated by the National Comprehensive Cancer Network (NCCN). In the remaining group of 33 laboratories with incorrect variant results, less correct classifications were acquired for the variants with strong clinical significance. Conclusions: Owing to numerous reasons, the interpretation of variants differed greatly, which might in turn lead to the inappropriate clinical care of patients with NSCLC. By analyzing the limitations of different databases used by laboratories, we integrated various types of databases with different levels of evidence to form a comprehensive and detailed variant interpretation pipeline, aiming to standardize the variant classification and provide accurate and sufficient therapeutic drug recommendation to clinicians for minimal-inappropriate therapeutic options.