Virological and Mitochondriopathogical Characteristics of the SARS-CoV-2 Omicron XBB.1.16 Spike.

The emergence of XBB.1.16 has gained rapid global prominence. Previous studies have elucidated that the infection of SARS-CoV-2 induces alterations in the mitochondrial integrity of host cells, subsequently influencing the cellular response to infection. In this study, we compared the differences in infectivity and pathogenicity between XBB.1.16 and the parental Omicron sublineages BA.1 and BA.2 and assessed their impact on host mitochondria. Our findings suggest that, in comparison with BA.1 and BA.2, XBB.1.16 exhibits more efficient spike protein cleavage, more efficient mediating syncytia formation, mild mitochondriopathy, and less pathogenicity. Altogether, our investigations suggest that, based on the mutation of key sites, XBB.1.16 exhibited enhanced infectivity but lower pathogenicity. This will help us to further investigate the biological functions of key mutation sites.

Non-tumor-related prognostic factors for immunotherapy-chemotherapy or immunotherapy alone as first-line in advanced non-small cell lung cancer (NSCLC).

Besides programmed death ligand 1 (PD-L1) expression, rapid, cost-effective and validated scores or models are critical for the prognosis and prediction of patients received immune checkpoint inhibitors (ICIs). In this retrospective study, 182 patients with NSCLC receiving ICIs from 2015 to 2022 were divided 1:1 into a training cohort and a validation cohort. We identified a score established by three factors and analyzed the prognostic implications by Kaplan-Meier approach (Log rank test) and time-dependent receiver operating characteristic (ROC) analyses. A non-tumor-related score (NTRS) was established that could be used as a prognostic factor (HR 2.260, 95% CI 1.559-3.276, P < 0.001 in training cohort; HR 2.114, 95% CI 1.493-2.994, P < 0.001 in validation cohort) and had a high time-dependent ROC for overall survival (OS) (AUC 0.670-0.782 in training cohort; AUC 0.682-0.841 in validation cohort). PD-L1 (1-49%) and NTRS (score = 0, 1, 2, 3) combination significantly improved the assessment of patients' OS and progress-free survival (PFS), which was statistically different in training cohorts (P < 0.001 for OS, 0.012 for PFS) and validation cohorts (P = 0.01 for OS, < 0.001 for PFS). The NTRS provided a better assessment of durable clinical benefit (DCB) compared to PD-L1 expression (P = 0.009 vs. 0.232 in training cohort; P = 0.004 vs. 0.434 in validation cohort). NTRS may help improve prognosis stratification of patients receiving ICIs in first-line NSCLC and may be combined with tumor-related parameters.

Expert Consensus on the Diagnosis and Treatment of NRG1/2 Gene Fusion Solid Tumors.

The fusion genes NRG1 and NRG2 , members of the epidermal growth factor (EGF) receptor family, have emerged as key drivers in cancer. Upon fusion, NRG1 retains its EGF-like active domain, binds to the ERBB ligand family, and triggers intracellular signaling cascades, promoting uncontrolled cell proliferation. The incidence of NRG1 gene fusion varies across cancer types, with lung cancer being the most prevalent at 0.19 to 0.27%. CD74 and SLC3A2 are the most frequently observed fusion partners. RNA-based next-generation sequencing is the primary method for detecting NRG1 and NRG2 gene fusions, whereas pERBB3 immunohistochemistry can serve as a rapid prescreening tool for identifying NRG1 -positive patients. Currently, there are no approved targeted drugs for NRG1 and NRG2 . Common treatment approaches involve pan-ERBB inhibitors, small molecule inhibitors targeting ERBB2 or ERBB3, and monoclonal antibodies. Given the current landscape of NRG1 and NRG2 in solid tumors, a consensus among diagnostic and treatment experts is proposed, and clinical trials hold promise for benefiting more patients with NRG1 and NRG2 gene fusion solid tumors.

The Developed Wave Cancellation Theory Contributing to Understand Wave Absorption Mechanism of ZIF Derivatives with Controllable Electromagnetic Parameters.

How to better understand the influence of electromagnetic parameters on the absorbing properties of electromagnetic wave absorbers (EMAs) is an essential prerequisite for further synthesis and development of high-performance EMAs. In this work, an improved wave cancellation theory is used as a guiding principle to prepare N-doped carbon-coated cobalt nanoparticles (Co@NC) using ZIF-8@ZIF-67 as the precursor, thus enabling controllable electromagnetic parameters by regulating the conduction loss and dipole polarization ability. The Co@NC generated by pyrolysis at 700 °C under H2 atmosphere presents an optimized absorption performance. Benefiting from developed wave cancellation theory, the thickness of the film can be accurately adjusted so that the difference between the amplitude of the reflected and transmitted electromagnetic waves is only 0.001 and the phase difference is 180.05°, thus achieving a minimum reflection loss (RLmin (dB)) of -64.0 dB. Meanwhile, a maximum effective absorption bandwidth of 5.4 GHz is achieved simultaneously attributing to its most suitable electromagnetic parameters. Accordingly, the current research based on wave cancellation theory significantly contributes to understand the relationships between electromagnetic parameters and wave absorption properties, therefore providing a theoretical insight into the further development of high-performance EMAs.

Hsa-LINC02418/mmu-4930573I07Rik regulated by METTL3 dictates anti-PD-L1 immunotherapeutic efficacy via enhancement of Trim21-mediated PD-L1 ubiquitination.

BACKGROUND: Limited response to programmed death ligand-1 (PD-L1)/programmed death 1 (PD-1) immunotherapy is a major hindrance of checkpoint immunotherapy in non-small cell lung cancer (NSCLC). The abundance of PD-L1 on the tumor cell surface is crucial for the responsiveness of PD-1/PD-L1 immunotherapy. However, the negative control of PD-L1 expression and the physiological significance of the PD-L1 inhibition in NSCLC immunotherapy remain obscure. METHODS: Bioinformatics analysis was performed to profile and investigate the long non-coding RNAs that negatively correlated with PD-L1 expression and positively correlated with CD8+T cell infiltration in NSCLC. Immunofluorescence, in vitro PD-1 binding assay, T cell-induced apoptosis assays and in vivo syngeneic mouse models were used to investigate the functional roles of LINC02418 and mmu-4930573I07Rik in regulating anti-PD-L1 therapeutic efficacy in NSCLC. The molecular mechanism of LINC02418-enhanced PD-L1 downregulation was explored by immunoprecipitation, RNA immunoprecipitation (RIP), and ubiquitination assays. RIP, luciferase reporter, and messenger RNA degradation assays were used to investigate the m6A modification of LINC02418 or mmu-4930573I07Rik expression. Bioinformatics analysis and immunohistochemistry (IHC) verification were performed to determine the significance of LINC02418, PD-L1 expression and CD8+T cell infiltration. RESULTS: LINC02418 is a negative regulator of PD-L1 expression that positively correlated with CD8+T cell infiltration, predicting favorable clinical outcomes for patients with NSCLC. LINC02418 downregulates PD-L1 expression by enhancing PD-L1 ubiquitination mediated by E3 ligase Trim21. Both hsa-LINC02418 and mmu-4930573I07Rik (its homologous RNA in mice) regulate PD-L1 therapeutic efficacy in NSCLC via Trim21, inducing T cell-induced apoptosis in vitro and in vivo. Furthermore, METTL3 inhibition via N6-methyladenosine (m6A) modification mediated by YTHDF2 reader upregulates hsa-LINC02418 and mmu-4930573I07Rik. In patients with NSCLC, LINC02418 expression is inversely correlated with PD-L1 expression and positively correlated with CD8+T infiltration. CONCLUSION: LINC02418 functions as a negative regulator of PD-L1 expression in NSCLC cells by promoting the degradation of PD-L1 through the ubiquitin-proteasome pathway. The expression of LINC02418 is regulated by METTL3/YTHDF2-mediated m6A modification. This study illuminates the underlying mechanisms of PD-L1 negative regulation and presents a promising target for improving the effectiveness of anti-PD-L1 therapy in NSCLC.

Expert consensus on the diagnosis and treatment of RET gene fusion non-small cell lung cancer in China.

The rearranged during transfection (RET) gene is one of the receptor tyrosine kinases and cell-surface molecules responsible for transmitting signals that regulate cell growth and differentiation. In non-small cell lung cancer (NSCLC), RET fusion is a rare driver gene alteration associated with a poor prognosis. Fortunately, two selective RET inhibitors (sRETi), namely pralsetinib and selpercatinib, have been approved for treating RET fusion NSCLC due to their remarkable efficacy and safety profiles. These inhibitors have shown the ability to overcome resistance to multikinase inhibitors (MKIs). Furthermore, ongoing clinical trials are investigating several second-generation sRETis that are specifically designed to target solvent front mutations, which pose a challenge for first-generation sRETis. The effective screening of patients is the first crucial step in the clinical application of RET-targeted therapy. Currently, four methods are widely used for detecting gene rearrangements: next-generation sequencing (NGS), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). Each of these methods has its advantages and limitations. To streamline the clinical workflow and improve diagnostic and treatment strategies for RET fusion NSCLC, our expert group has reached a consensus. Our objective is to maximize the clinical benefit for patients and promote standardized approaches to RET fusion screening and therapy.

Chinese expert consensus on the diagnosis and treatment of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a malignant tumor originating from the pleura, and its incidence has been increasing in recent years. Due to the insidious onset and strong local invasiveness of MPM, most patients are diagnosed in the late stage and early screening and treatment for high-risk populations are crucial. The treatment of MPM mainly includes surgery, chemotherapy, and radiotherapy. Immunotherapy and electric field therapy have also been applied, leading to further improvements in patient survival. The Mesothelioma Group of the Yangtze River Delta Lung Cancer Cooperation Group (East China LUng caNcer Group, ECLUNG; Youth Committee) developed a national consensus on the clinical diagnosis and treatment of MPM based on existing clinical research evidence and the opinions of national experts. This consensus aims to promote the homogenization and standardization of MPM diagnosis and treatment in China, covering epidemiology, diagnosis, treatment, and follow-up.

Pneumatosis intestinalis post steroid use in a patient with immune-related adverse events: Case report, literature review and FAERS analysis.

Introduction: The accurate diagnosis of pneumatosis intestinalis (PI) is increasing despite patients' limited identification of etiologic factors. Recently a patient with lung squamous carcinoma who developed pneumatosis intestinalis following methylprednisolone administration for immune-related adverse events was treated at our hospital. Subsequent a literature review and an analysis of the FDA Adverse Event Reporting System (FAERS) database enabled the identification of additional cases of pneumatosis intestinalis. Methods: A literature review of the MEDLINE/PubMed and Web of Science Core Collection databases using standard pneumatosis intestinalis search terms to identify published cases of immune checkpoint inhibitors (ICIs) or steroids causing pneumatosis intestinalis were performed. A separate retrospective pharmacovigilance study of FAERS enabled the extraction of unpublished cases of pneumatosis intestinalis between the first quarter of 2005 and the third quarter of 2022. Disproportionality and Bayesian analyses were performed to identify signal detection in reported odds ratios, proportional reporting ratios, information components, and empirical Bayesian geometric means. Results: Ten case reports of steroid-related pneumatosis intestinalis were retrieved from six published studies. The implicated drug therapies included pre-treatment with steroids before chemotherapy, combination therapy with cytotoxic agents and steroids, and monotherapy with steroids. In the FAERS pharmacovigilance study, 1,272 cases of immune checkpoint inhibitors or steroid-related pneumatosis intestinalis were incidentally reported. The signal detected in five kinds of immune checkpoint inhibitors and six kinds of steroids implied a positive correlation between the drugs and adverse events. Conclusion: Steroids might be the etiologic factors in the current case of pneumatosis intestinalis. Reports supporting the role of steroids in suspected cases of pneumatosis intestinalis can be found in literature databases and the FAERS database. Even so, as documented in FAERS, immune checkpoint inhibitors-induced pneumatosis intestinalis should not be excluded.

Chinese expert consensus on the diagnosis and treatment of thymic epithelial tumors.

Thymic epithelial tumors (TETs) are a relatively rare type of thoracic tumor, accounting for less than 1% of all tumors. The incidence of TETs is about 3.93/10000 in China, slightly higher than that of European and American countries. For resectable TETs, complete surgical resection is recommended. Radiotherapy or chemotherapy may be used as postoperative adjuvant treatment. Treatment for advanced, unresectable TETs consist mainly of radiotherapy and chemotherapy, but there is a lack of standard first- and second-line treatment regimens. Recently, targeted therapies and immune checkpoint inhibitors have shown promising outcomes in TETs. Based on the currently available clinical evidences and the opinions of the national experts, the Thymic Oncology Group of Yangtze River Delta Lung Cancer Cooperation Group (East China LUng caNcer Group, ECLUNG; Youth Committee) established this Chinese expert consensus on the clinical diagnosis and treatment of TETs, covering the epidemiology, diagnosis, treatment, prognosis and follow-up of TETs.

Chinese expert consensus on the diagnosis and treatment of HER2-altered non-small cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) possesses tyrosine kinase activity and participates in cell growth, differentiation and migration, and survival. Its alterations, mainly including mutations, amplifications, and overexpression are associated with poor prognosis and are one of the major drivers in non-small cell lung cancer (NSCLC). Several clinical trials had been investigating on the treatments of HER2-altered NSCLC, including conventional chemotherapy, programmed death 1 (PD-1) inhibitors, tyrosine kinase inhibitors (TKIs) and antibody-drug conjugates (ADCs), however, the results were either disappointing or encouraging, but inconsistent. Trastuzumab deruxtecan (T-DXd) was recently approved by the Food and Drug Administration as the first targeted agent for treating HER2-mutant NSCLC. Effective screening of patients is the key to the clinical application of HER2-targeted agents such as TKIs and ADCs. Various testing methods are nowadays available, including polymerase chain reaction (PCR), next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), etc. Each method has its pros and cons and should be reasonably assigned to appropriate patients for diagnosis and guiding treatment decisions. To help standardize the clinical workflow, our expert group reached a consensus on the clinical management of HER2-altered NSCLC, focusing on the diagnosis and treatment strategies.

Chinese expert consensus on the multidisciplinary management of pneumonitis associated with immune checkpoint inhibitor.

Immune checkpoint inhibitors (ICIs) have successfully treated a number of different types of cancer, which is of great significance for cancer treatment. With the widespread use of ICIs in clinical practice, the increasing checkpoint inhibitor pneumonia (CIP) will be a challenge to clinicians. To guide the diagnosis and treatment of CIP, we conducted in-depth discussions based on the latest evidence, forming a consensus among Chinese experts on the multidisciplinary management of CIP.

Expert consensus on the diagnosis and treatment of NTRK gene fusion solid tumors in China.

Gene fusions can drive tumor development for multiple types of cancer. Currently, many drugs targeting gene fusions are being approved for clinical application. At present, tyrosine receptor kinase (TRK) inhibitors targeting neurotrophic tyrosine receptor kinase (NTRK) gene fusions are among the first "tumor agnostic" drugs approved for pan-cancer use. Representative TRK inhibitors, including larotrectinib and entrectinib, have shown high efficacy for many types of cancer. At the same time, several second-generation drugs designed to overcome first-generation drug resistance are undergoing clinical development. Due to the rarity of NTRK gene fusions in common cancer types and technical issues regarding the complexity of fusion patterns, effectively screening patients for TRK inhibitor treatment in routine clinical practice is challenging. Different detection methods including immunohistochemistry, fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and (DNA and/or RNA-based) next-generation sequencing have pros and cons. As such, recommending suitable tests for individual patients and ensuring the quality of tests is essential. Moreover, at present, there is a lack of systematic review for the clinical efficacy and development status of first- and second-generation TRK inhibitors. To resolve the above issues, our expert group has reached a consensus regarding the diagnosis and treatment of NTRK gene fusion solid tumors, aiming to standardize clinical practice with the goal of benefiting patients with NTRK gene fusions treated with TRK inhibitors.

Morphology-Controlled Fabrication Strategy of Hollow Mesoporous Carbon Spheres@f-Fe2O3 for Microwave Absorption and Infrared Stealth.

The design and development of radar--infrared compatible stealth materials are challenging in the field of broadband absorption due to the contradiction of stealth requirements and mechanisms in different frequency bands. However, hollow structures show great promise for multispectral stealth because they can lengthen the attenuation path of electromagnetic waves (EMWs) for microwave absorption, interrupt the continuity of heat-transport channels, and lower the thermal conductivity to realize infrared stealth. Here, a new morphological fabrication strategy has been developed to efficiently prepare compatible stealth nanomaterials. In a specific hydrothermal process, the confined growth of flake α-Fe2O3 (f-Fe2O3) outside of hollow mesoporous carbon spheres (HMCS) is achieved using NH3·H2O as a shape-controlled reagent. The introduction of f-Fe2O3 helps to lower infrared emissivity and improve high-frequency impedance matching, which depends on the stable dielectric property of the specific flake shape. Moreover, the size of f-Fe2O3 can be regulated by changing the constituent proportion in the hydrothermal suspension to obtain excellent performance. The minimum reflection loss (RL) of the HMCS@f-Fe2O3-6 composite is -34.16 dB at 2.4 mm, and the effective absorption bandwidth (EAB) reaches 4.8 GHz. Furthermore, the lowest emissivities of the HMCS@f-Fe2O3-6-20 wt %/polyetherimide (PEI) film in the 3-5 and 8-14 µm infrared wavebands are 0.212 and 0.508, respectively. These discoveries may pave the way for the development of radar-infrared compatible stealth materials from the perspective of microstructural design.

Case Report: Complete Remission With Anti-PD-1 and Anti-VEGF Combined Therapy of a Patient With Metastatic Primary Splenic Angiosarcoma.

Primary splenic angiosarcoma (PSA) is a rare malignancy with poor prognosis. At present, little study is available on immunotherapy in PSA. Here, we report a case of a patient with metastatic PSA who was treated with programmed death-1 (PD-1) inhibitors and vascular endothelial growth factor (VEGF) tyrosine kinase inhibitors combined therapy and achieved complete response (CR). The patient was a 57-year-old woman with three liver metastases. She was treated with seven cycles of toripalimab plus anlotinib. Programmed death-ligand 1 (PD-L1) immunohistochemistry and next-generation sequencing was performed, and the PD-L1 tumor proportion score was 75%. Finally, she achieved CR after six cycles of the combined therapy regimen. No serious adverse events were detected. To the best of our knowledge, this is the first clinical evidence that anti-PD-1 plus anti-VEGF therapy might be a promising option for patients with metastatic PSA. However, more clinical trials are needed to verify this conclusion.

[Blood-based Biomarkers in the Immune Checkpoint Inhibitor Treatment in 
Non-small Cell Lung Cancer].

Immune checkpoint inhibitors (ICI) have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). Biomarkers are essential for guiding precision immunotherapy. Tissue-based programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB) are currently widely used biomarkers for selecting patients for immunotherapy. However, tissue specimens are often difficult to reach and couldn't overcome spatial and temporal heterogeneity. Blood biomarkers offer an alternative non-invasive solution that could provide a complete insight on patient's immune status and tumor as well, and show their potential in predicting the outcome as well as in monitoring response to immunotherapy. In this article, we summarize current knowledge on blood biomarkers in NSCLC patients treated with ICI, and we hope to provide more references for development of novel biomarkers.
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Establishment and validation of a novel droplet digital PCR assay for ultrasensitive detection and dynamic monitoring of EGFR mutations in peripheral blood samples of non-small-cell lung cancer patients.

BACKGROUND: Droplet digital PCR (ddPCR)-based blood detection of EGFR mutations plays significant roles in the individualized therapy of non-small-cell lung cancer (NSCLC) patients. However, a standard assay that is approved by health authorities is still lacking. Additionally, the proper application of this method in clinical settings also needs further investigation. METHODS: The performance of a newly established ddPCR assay was first evaluated using reference samples and then validated by comparing this method with the amplification refractory mutation system (ARMS) using cell-free DNA (cfDNA) in patients' peripheral blood. Further, the correlation between dynamic quantification of EGFR mutation in the patients and their clinical outcome of tyrosine kinase inhibitors (TKIs) therapy was investigated. RESULTS: A total of 77 patients were included, with 50 in the test group and 27 in the validation group. According to the results of the reference samples and the blood samples in the test group, the cut-off value for patient detection was proposed as mutation rate ≥ 0.1% (total copy number of cfDNA ≥ 1000) or at least one copy of mutation DNA was detected (total copy number of cfDNA < 1000). With this criterion, superior sensitivity of our assay to that of ARMS was observed (P = 0.002 for Ex19Del & L858R and P < 0.001 for T790M). The dynamic quantification of EGFR mutations during TKI therapy indicated that an increase in mutation abundance was correlated with resistance, while a decline was associated with response. Notably, a rebound in mutation abundance during chemotherapy may indicate a desirable chance for TKI re-treatment. CONCLUSION: The novel ddPCR assay showed superior sensitivity in the detection of EGFR mutation in blood. The dynamic quantification of EGFR mutations by this assay would greatly facilitate the administration of TKI therapy, including the monitoring of resistance and response, as well as cohort screening for retreatment.

Simultaneous Detection of Exosomal Membrane Protein and RNA by Highly Sensitive Aptamer Assisted Multiplex-PCR.

Exosomes, which are 30-150 nm extracellular vesicles containing a variety of biomolecules (i.e., proteins, RNA, DNA, and lipids), have emerged as important analytes in liquid biopsy. Although exosome detection offers a valuable chance to understand disease status in a multidimensional manner, comprehensive analysis of different exosomal biomolecules with limited specimens remains a challenge. Herein, we introduced an aptamer-based PCR (polymerase chain reaction) platform and realized highly sensitive detection of CD63 protein positive exosomes as low as 50 particles/µL. More importantly, simultaneous analysis of exosomal RNA and surface protein is available using this method. In a proof of concept experiment, simultaneous detection of two immunotherapy-related biomarkers, programmed death-ligand 1 (PD-L1) protein and indoleamine 2,3-dioxygenase 1 (IDO1) mRNA, was demonstrated with clinical samples. Our method provides a strategy for the comprehensive analysis of exosomes, with the advantages of high sensitivity and practicability.

Exploratory study on application of MALDI­TOF­MS to detect serum and urine peptides related to small cell lung carcinoma.

Matrix­assisted laser desorption/ionization time­of­flight mass spectrometry (MALDI­TOF­MS) was employed to analyze differential serum and urine peptides in patients with small cell lung cancer (SCLC) and healthy individuals, and SCLC diagnostic classification models were constructed. Serum and urine samples from 72 patients with SCLC, age­ and gender­matched with 72 healthy individuals, were divided into training and testing sets in a 3:1 ratio. Serum and urine peptides were extracted using copper ion­chelating nanomagnetic beads, and mass spectra were obtained using MALDI­TOF­MS. Peptide spectra for the training set were analyzed, and the classification model was constructed using ClinProTools (CPT). The testing set was used for blinded model validation. For training­set sera, 122 differential peptide signal peaks with a mass of 0.8­10 kDa were observed, and 19 peptides showed significantly different expression [P<0.0005; area under curve (AUC) ≥0.80]. CPT screened 5 peptide peaks (0.8114, 0.83425, 1.86655, 4.11133 and 5.81192 kDa) to construct the classification model. The testing set was used for the blinded validation, which had 95.0% sensitivity and 90.0% specificity. For the training­set urine, 132 differential peptide signal peaks with m/z ratios of 0.8­10 kDa were observed, and 8 peptides had significantly different expression (P<0.0005; AUC ≥0.80). Then, 5 peaks (1.0724, 2.37692, 2.7554, 4.75475 and 4.7949 kDa) were used for classification model construction. The testing set was used for 36 blinded validation, which had 85.0% sensitivity and 80.0% specificity. Among the differential peptides, 3 had the same significant peaks at 2.3764, 0.8778 and 0.8616 kDa, identified as fibrinogen α, glucose­6­phosphate isomerase and cyclin­dependent kinase­1, respectively. The present study highlighted the differences that exist in serum and urine peptides between patients with SCLC and healthy individuals. Serum and urine peptide diagnostic classification models could be constructed using MALDI­TOF­MS, and showed high sensitivity and specificity.

Accurate Classification of Non-small Cell Lung Cancer (NSCLC) Pathology and Mapping of EGFR Mutation Spatial Distribution by Ambient Mass Spectrometry Imaging.

Objectives: Tumor pathology examination especially epidermal growth factor receptor (EGFR) mutations molecular testing has been integral part of lung cancer clinical practices. However, the EGFR mutations spatial distribution characteristics remains poorly investigated, which is critical to tumor heterogeneity analysis and precision diagnosis. Here, we conducted an exploratory study for label-free lung cancer pathology diagnosis and mapping of EGFR mutation spatial distribution using ambient mass spectrometry imaging (MSI). Materials and Methods: MSI analysis were performed in 55 post-operative non-small cell lung cancer (NSCLC) tumor and paired normal tissues to distinguish tumor from normal and classify pathology. We then compared diagnostic sensitivity of MSI and ADx-amplification refractory mutation system (ARMS) for the detection of EGFR mutation in pathological confirmed lung adenocarcinoma (AC) and explored EGFR mutations associated biomarkers to depict EGFR spatial distribution base on ambient MSI. Results: Of 55 pathological confirmed NSCLC, MSI achieved a diagnostic sensitivity of 85.2% (23/27) and 82.1% (23/28) for AC and squamous cell carcinoma (SCC), respectively. Among 27 AC, there were 17 EGFR-wild-type and 10 EGFR-mutated-positive samples detected by ARMS, and MSI achieved a diagnostic sensitivity of 82.3% (14/17) and 80% (8/10) for these two groups. Several phospholipids were specially enriched in AC compared with SCC tissues, with the higher ions intensity of phospholipids in EGFR-mutated-positive compared with EGFR-wild-type AC tissues. We also found EGFR mutations distribution was heterogeneous in different regions of same tumor by multi-regions ARMS detection, and only the regions with higher ions intensity of phospholipids were EGFR-mutated-positive. Conclusion: MSI method could accurately distinguish tumor pathology and subtypes, and phospholipids were reliable EGFR mutations associated biomarkers, phospholipids imaging could intuitively visualize EGFR mutations spatial distribution, may facilitate our understanding of tumor heterogeneity.

Aptamer-based fluorescence polarization assay for separation-free exosome quantification.

Tumor-derived exosomes have emerged as promising cancer biomarkers and attracted increasing interest in non-invasive cancer diagnosis and treatment monitoring. However, the identification and quantification of exosomes in clinical samples such as blood remains challenging due to the difficulty in trade-off between recognition specificity and isolation efficiency. Here we have developed an aptamer-based fluorescence polarization assay for exosome quantification, which is a separation-free, amplification-free and sensitive approach enabling direct quantification of exosomes in human plasma. While the key specificity of this assay is based on the aptamer's inherent affinity to membrane proteins on exosomes, exosomes' inherent huge mass/volume acts as mass-based fluorescence polarization amplifier. Our assay allows quantitative analysis of exosomes in the range of 5 × 102 to 5 × 105 particles per µL with a detect limitation of 500 particles per µL for the cell line deprived exosomes. The total assay time is about 30 min with just one mix-and-read step to achieve high sensitivity. We have also demonstrated quantification of exosomes from lung cancer patients and healthy donors in clinical samples. This work describes a new and simple liquid biopsy assay to directly detect exosomes in the biological matrix, which facilitates cancer diagnosis and therapy monitoring.