Unraveling Mechanism for Microstructure Engineering toward High-Capacity Nickel-Rich Cathode Materials.

Microstructural engineering on nickel-rich layered oxide (NRLO) cathode materials is considered a promising approach to increase both the capacity and lifespan of lithium-ion batteries by introducing high valence-state elements. However, rational regulation on NRLO microstructures based on a deep understanding of its capacity enhancement mechanism remains challenging. Herein for the first time, it is demonstrated that an increase of 14 mAh g-1 in reversible capacity at the first cycle can be achieved via tailoring the micro and nano structure of NRLO through introducing tungsten. Aberration-corrected scanning transmission electron microscopy (STEM) characterization reveals that the formation of a modified microstructure featured as coherent spinel twin boundaries. Theoretical modeling and electrochemical investigations further demonstrate that the capacity increase mechanism is related to such coherent spinel twin boundaries, which can lower the Li+ diffusion barrier and thus allow more Li+ to participate in deeper phase transitions. Meanwhile, the surface and grain boundaries of NRLOs are found to be modified by generating a dense and uniform LiWxOy phase, which further extends its cycle life by reducing side reactions with electrolytes. This work enables a comprehensive understanding of the capacity-increased mechanism and endows the remarkable potential of microstructural engineering for capacity- and lifespan-increased NRLOs.

An ultrasensitive method for detecting mutations from short and rare cell-free DNA.

Circulating tumor DNA (ctDNA) was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. We developed the One-PrimER Amplification (OPERA) system and examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 bp, OPERA was capable of detecting mutations as low as 0.0025% VAF, while CAPP-Seq only detected mutations of >0.03% VAF. Both single nucleotide variant and insertion/deletion can be detected by OPERA. In synthetic fragments as short as 80 bp with low VAF (0.03%-0.1%), the detection sensitivity of OPERA was significantly higher compared to that of droplet digital polymerase chain reaction. The error rate was 5.9×10-5 errors per base after de-duplication in plasma samples collected from healthy volunteers. By suppressing "single-strand errors", the error rate can be further lowered by >5 folds in EGFR T790M hotspot. In plasma samples collected from lung cancer patients, OPERA detected mutations in 57.1% stage I patients with 100% specificity and achieved a sensitivity of 30.0% in patients with tumor volume of less than 1 cm3. OPERA can effectively detect mutations in rare and highly-fragmented DNA.

A novel microRNA-182/Interleukin-8 regulatory axis controls osteolytic bone metastasis of lung cancer.

Bone metastasis is one of the main complications of lung cancer and most important factors that lead to poor life quality and low survival rate in lung cancer patients. However, the regulatory mechanisms underlying lung cancer bone metastasis are still poor understood. Here, we report that microRNA-182 (miR-182) plays a critical role in regulating osteoclastic metastasis of lung cancer cells. We found that miR-182 was significantly upregulated in both bone-metastatic human non-small cell lung cancer (NSCLC) cell line and tumor specimens. We further demonstrated that miR-182 markedly enhanced the ability of NSCLC cells for osteolytic bone metastasis in nude mice. Mechanistically, miR-182 promotes NSCLC cells to secrete Interleukin-8 (IL-8) and in turn facilitates osteoclastogenesis via activating STAT3 signaling in osteoclast progenitor cells. Importantly, systemically delivered IL-8 neutralizing antibody inhibits NSCLC bone metastasis in nude mice. Collectively, our findings identify the miR-182/IL-8/STAT3 axis as a key regulatory pathway in controlling lung cancer cell-induced osteolytic bone metastasis and suggest a promising therapeutic strategy that targets this regulatory axis to interrupt lung cancer bone metastasis.

Comparative analysis of QS3D versus droplet digital PCR for quantitative measures of EGFR T790M mutation from identical plasma.

Objectives: The capacity of QuantStudio™ 3D (QS3D) and droplet digital PCR (dPCR) for the detection of plasma Epidermal Growth Factor Receptor (EGFR) mutations have been widely reported. Few comparative studies on the quantitative test of the identical DNA material, however, are carried out. Here we compared the performance of the two methods in detecting EGFR T790M mutation in cell-free DNA (cfDNA) from the same lung cancer patients. Methods: We recruited 72 non-small cell lung cancer (NSCLC) patients who initially respond to tyrosine kinase inhibitor treatment but subsequently developed resistance. Two tubes of 10mL anticoagulant blood were collected and cfDNA was isolated from plasma. Identical cfDNA samples were analyzed for T790M mutation using QS3D and droplet dPCR in parallel. Results: T790M mutation was detected in 15 and 21 cfDNA samples by QS3D and droplet digital PCR, respectively. The 6 discordant samples showed low mutation abundance (∼0.1%) and the discrepancy is caused by the stricter threshold settings for QS3D dPCR. The overall agreement between the two methods was 91.7% (66/72). The median allele frequencies for QS3D dPCR and droplet dPCR to detect T790M mutation was 2.01% and 2.62%, respectively. There was no significance in mutation abundance detected by both methods. Both methods are highly correlated with allele frequencies and copy numbers in T790M wild type and mutant, with R2 of 0.98, 0.92 and 0.95, respectively. Conclusion: Our study demonstrated that QS3D dPCR are highly consistent with droplet PCR for quantitative determination of EGFR T790M mutation in plasma cfDNA.

Integrative Serum Metabolic Fingerprints Based Multi-Modal Platforms for Lung Adenocarcinoma Early Detection and Pulmonary Nodule Classification.

Identification of novel non-invasive biomarkers is critical for the early diagnosis of lung adenocarcinoma (LUAD), especially for the accurate classification of pulmonary nodule. Here, a multiplexed assay is developed on an optimized nanoparticle-based laser desorption/ionization mass spectrometry platform for the sensitive and selective detection of serum metabolic fingerprints (SMFs). Integrative SMFs based multi-modal platforms are constructed for the early detection of LUAD and the classification of pulmonary nodule. The dual modal model, metabolic fingerprints with protein tumor marker neural network (MP-NN), integrating SMFs with protein tumor marker carcinoembryonic antigen (CEA) via deep learning, shows superior performance compared with the single modal model Met-NN (p < 0.001). Based on MP-NN, the tri modal model MPI-RF integrating SMFs, tumor marker CEA, and image features via random forest demonstrates significantly higher performance than the clinical models (Mayo Clinic and Veterans Affairs) and the image artificial intelligence in pulmonary nodule classification (p < 0.001). The developed platforms would be promising tools for LUAD screening and pulmonary nodule management, paving the conceptual and practical foundation for the clinical application of omics tools.

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells.

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based EGFR p.T790M in non-small-cell lung cancer patients.

BACKGROUND: Developing liquid biopsy technology with higher sensitivity and specificity especially for low-frequency mutations remains crucial. This study demonstrated superior performance of the newly developed digital PCR (dPCR) kit for ctDNA-based EGFR p.T790M detection in metastatic non-small-cell lung cancer (NSCLC) against ARMS-PCR. METHODS: This large-scale, multi-centered diagnostic study recruited 1,045 patients including 1,029 patients diagnosed with advanced NSCLC and 16 patients with specific samples between April 1st 2018 and November 30th 2019. EGFR p.T790M in plasma samples from mNSCLC patients were tested using dPCR with ADx-ARMS PCR and Cobas® EGFR Mutation Test V2 as comparator assays to confirm cut-off value for dPCR and evaluate its performance against ARMS-PCR-based assays. Efficacy was evaluated for patients with EGFR p.T790M detected by dPCR or ARMS-PCR, who underwent Osimertinib treatment. RESULTS: The sensitivity, specificity, and concordance of dPCR against ADx-ARMS PCR was 98.15%, 88.66% and 90.16%, respectively for 1,026 plasma samples. Additional 9.26% patients were detected positive by dPCR. The majority of those samples had a mutation allele frequency between 0.1% and 1%. In 45 paired tissue and plasma samples, the sensitivity improved from 30.77% to 53.85% by dPCR with the specificity over 90%. The response of Osimertinib in 74 EGFR p.T790M-positive patients detected by dPCR, including 26 determined as negative by ARMS-PCR, were evaluated to have an ORR of 44.59% and a DCR of 90.54%. CONCLUSIONS: dPCR is a sensitive and accurate tool for ctDNA-based EGFR p.T790M detection due to its significantly improved sensitivity without compromising specificity, and dPCR is equivalent to ARMS-PCR as a companion diagnostic tool while benefiting more patients under Osimertinib treatment. TRIAL REGISTRATION: Chinese Clinical Trial Registry identifier: ChiCTR2100043147.

Non-small cell lung cancer cell-derived exosomal miR-17-5p promotes osteoclast differentiation by targeting PTEN.

Aberrant activity of bone resorbing osteoclasts plays a key role in the development of osteoporosis and cancer bone metastasis. The identification of novel and specific targets will be helpful for the development of new therapeutic strategies for bone metastasis in lung cancer. Herein, we examined microRNAs in tumor cell-derived exosomes to investigate the communication between the bone environment and tumor cells. TCGA database analysis showed that the level of miR-17-5p increased in non-small cell lung cancer tissues compared with non-tumor tissues. To investigate the function of exosomes in inducing osteoclastogenesis, osteoclast precursors were incubated with exosomes isolated from non-small cell lung cancer cell line, as well as receptor activator of NF-KB ligand and M-CSF to induce osteoclastogenesis. We found that exosomal miR-17-5p is upregulated in a non-small cell lung cancer cell line with bone metastasis compared with the original cell line. Overexpression of miR-17-5p enhanced the osteoclastogenesis of RAW264.7 cells. PTEN was identified as a direct target of miR-17-5p and showed negative effects on osteoclastogenesis. Importantly, treatment of LY294002 (an inhibitor of the PI3K/Akt pathway) attenuated miR-17-5p-mediated osteoclastogenesis effects. Taken together, our findings demonstrated that miR-17-5p promotes osteoclastogenesis through the PI3K/Akt pathway via targeting PTEN in lung cancer.

Performance comparison of commercial kits for isolating and detecting circulating tumor DNA.

Circulating tumor DNA (ctDNA), a fraction of cell-free DNA (cfDNA) in the circulatory system, is released from tumor cells and thus carries tumor-specific genetic signatures. Using blood-derived ctDNA to detect somatic mutations has shown great value in guiding cancer targeted therapy. Isolation and detection efficiencies are the key factors affecting the performance of ctDNA detection. To optimize and standardize our clinical practice, in this study, we analyzed the isolation efficiency of four commercial cfDNA purification kits: QIAamp circulating nucleic acid kit, AmoyDx® Circulating DNA kits, Microdiag® circulating DNA isolation kit, and MagMAX cell-free DNA isolation kit; and the detection efficiency of two mainstream domestic EGFR gene mutation detection kits: MicroDiag EGFR gene mutation detection kit and Fluorometric real-time PCR Detection Kit for the analysis of EGFR gene mutations. Reference materials and plasma samples collected from lung cancer patients and healthy volunteers were used for the analysis. Our results showed that QIAamp circulating nucleic acid kit and Microdiag® circulating DNA kit had the highest recovery rate (up to 21.25 ng/mL) for short DNA fragments of about 173 bp which is the peak length of ctDNA. For ctDNA detection, the MicroDiag® EGFR gene mutation detection kit showed the highest detection rate and sensitivity for detecting EGFR mutations at a mutant frequency of 0.5%. This work provides a reliable choice of commercial kits for the clinical application of ctDNA.

Simplified ARCHITECT microfluidic chip through a dual-flip strategy enables stable and versatile tumoroid formation combined with label-free quantitative proteomic analysis.

Recent years, microfluidic three-dimensional (3D) tumor culture technique has made great progress in tumor microenvironment simulation and drug screening. Meanwhile, as their functionality and complexity increase, it is more difficult for current chip models to selectively collect specific-layer cells from tumoroids for further analysis. Moreover, a simplified and robust method for tumoroid formation with highly consistent size and repeatable 3D morphology is relatively ncessary. Here, we report an ARCHITECT (ARtificial CHIp for Tumor Enables Confocal Topography observation) chip, through a dual-flip strategy to implement straightforward tumoroid establishment. This platform guarantees stable batch-to-batch tumoroids formation and allows high resolution confocal imaging. Moreover, an initial cell density as low as 65 cells per chamber is efficient to deliver a tumoroid. With this ARCHITECT chip, different-layer cells of interest could be collected from tumoroid for label-free quantitative (LFQ) proteomic analysis. For application demonstration, we mainly verified this platform for lung carcinoma (A549) tumoroid construction and proteomic analysis at out layer. Our data indicate that the out-layer cells of A549 tumoroid show extensively distinct proteomic expressions compared to two-dimensional cultured A549 cells. The up-regulated proteins are mainly related to tumorigenicity, proliferation and metastasis. And the differentially expressed proteins are mainly relevant to lipid metabolism pathway which is essential to tumor progression and proliferation. This platform provides a simplified yet robust technique to connect microfluidic tumoroid construction and LFQ proteomic analysis. The simplicity of this technique should open the way to numerous applications such as discovering the innovative targets for cancer treatment, and studying the mophological and proteomic heterogeneity of different-layer cells across the tumoroid.

Label-free Separation of Circulating Tumor Cells Using a Self-Amplified Inertial Focusing (SAIF) Microfluidic Chip.

Circulating tumor cells (CTCs) are rare cells existing in the bloodstream with a relatively low number, which facilitate as a predictor of cancer progress. However, it is difficult to obtain highly purified intact CTCs with desired viability due to the low percentage of CTCs among blood cells. In this work, we demonstrate a novel self-amplified inertial focused (SAIF) microfluidic chip that enables size-based, high-throughput, label-free separation of CTCs from a patient's blood. The SAIF chip introduced in this study demonstrated the feasibility of an extremely narrow zigzag channel (with 40 µm channel width) connected with two expansion regions to effectively separate different-sized cells with amplified separation distance. The chip performance was optimized with different-sized polystyrene (PS) particles and blood cells spiked with three different types of cancer cells. The separation efficiencies for blood cells and spiked cancer cells are higher than 80%. Recovery rates of cancer cells were tested by spiking 1500 lung cancer cells (A549), breast cancer cells (MCF-7), and cervical cancer cells (HeLa) separately to 3 mL 0.09% saline with 3 × 106 white blood cells (WBCs). The recovery rates for larger cells (MCF-7 and HeLa) were 79.1 and 85.4%, respectively. Viabilities of the cells harvested from outlets were all higher than 97% after culturing for 24, 48, and 72 h. The SAIF chip performance was further confirmed using the real clinical patient blood samples from four lung cancer patients. Theoretical force balance analysis in physics, computational simulations, and experimental observations indicate that the SAIF chip is simple but effective, and high-throughput separation CTCs can be readily achieved without complex structures.

Machine learning of serum metabolic patterns encodes early-stage lung adenocarcinoma.

Early cancer detection greatly increases the chances for successful treatment, but available diagnostics for some tumours, including lung adenocarcinoma (LA), are limited. An ideal early-stage diagnosis of LA for large-scale clinical use must address quick detection, low invasiveness, and high performance. Here, we conduct machine learning of serum metabolic patterns to detect early-stage LA. We extract direct metabolic patterns by the optimized ferric particle-assisted laser desorption/ionization mass spectrometry within 1 s using only 50 nL of serum. We define a metabolic range of 100-400 Da with 143 m/z features. We diagnose early-stage LA with sensitivity~70-90% and specificity~90-93% through the sparse regression machine learning of patterns. We identify a biomarker panel of seven metabolites and relevant pathways to distinguish early-stage LA from controls (p < 0.05). Our approach advances the design of metabolic analysis for early cancer detection and holds promise as an efficient test for low-cost rollout to clinics.

High Expression of Long Noncoding RNA PCNA-AS1 Promotes Non-Small-Cell Lung Cancer Cell Proliferation and Oncogenic Activity via Upregulating CCND1.

Accumulating evidences showed that aberrantly expressed long noncoding RNAs (lncRNAs) have critical roles in many cancers. However, the expression and roles of a poorly studied lncRNA PCNA-AS1 in non-small-cell lung cancer (NSCLC) remain unknown. In this study, we investigated the expression, clinical significance, biological roles, and functional mechanism of PCNA-AS1 in NSCLC. Our results showed that PCNA-AS1 was upregulated in NSCLC tissues and cell lines, and correlated with TNM stages. Functional experiments showed that overexpression of PCNA-AS1 promoted NSCLC cell proliferation and cell cycle progression. Depletion of PCNA-AS1 inhibited NSCLC cell proliferation and cell cycle progression, and also inhibited NSCLC tumor growth in vivo. Mechanistically, we found that PCNA-AS1 upregulated CCND1 expression. The expression of PCNA-AS1 was positively correlated with that of CCND1 in NSCLC tissues. Moreover, depletion of CCND1 abrogated the oncogenic roles of PCNA-AS1 in NSCLC. In conclusion, highly expressed PCNA-AS1 promotes NSCLC cell proliferation and oncogenic activity via upregulating CCND1. Our results imply that PCNA-AS1 might serve as a therapeutic target for NSCLC.

Nanotechnology Strategies for the Analysis of Circulating Tumor DNA: A Review.

Circulating tumor DNA (ctDNA) describes the fragmented DNA released from tumor cells into the blood. The ctDNA may have the same genetic changes as the primary tumor. Currently, ctDNA has become a popular biomarker for diagnosis, treatment, real-time clinical response monitoring, and prognosis, for solid tumors. Detection of ctDNA is minimally invasive, and repeat sampling can easily be performed. However, due to its low quality and short DNA fragment length, ctDNA detection still faces challenges and requires highly sensitive analytical techniques. Recently, liquid biopsies for the analysis of circulating tumor cells (CTCs) and circulating tumor-derived exosomes have been studied, and nanotechnology techniques have rapidly developed. Compared to traditional analytical methods, these nanotechnology-based platforms have the advantages of sensitivity, multiplex detection, simplicity, miniaturization, and automation, which support their potential use in clinical practice. This review aims to discuss the recent nanotechnological strategies for ctDNA analysis and the design of reliable techniques for ctDNA detection and to identify the potential clinical applications.

Histone-Related Genes Are Hypermethylated in Lung Cancer and Hypermethylated HIST1H4F Could Serve as a Pan-Cancer Biomarker.

Lung cancer is the leading cause of cancer-related deaths worldwide. Cytologic examination is the current "gold standard" for lung cancer diagnosis, however, this has low sensitivity. Here, we identified a typical methylation signature of histone genes in lung cancer by whole-genome DNA methylation analysis, which was validated by The Cancer Genome Atlas (TCGA) lung cancer cohort (n = 907) and was further confirmed in 265 bronchoalveolar lavage fluid samples with specificity and sensitivity of 96.7% and 87.0%, respectively. More importantly, HIST1H4F was universally hypermethylated in all 17 tumor types from TCGA datasets (n = 7,344), which was further validated in nine different types of cancer (n = 243). These results demonstrate that HIST1H4F can function as a universal-cancer-only methylation (UCOM) marker, which may aid in understanding general tumorigenesis and improve screening for early cancer diagnosis. SIGNIFICANCE: These findings identify a new biomarker for cancer detection and show that hypermethylation of histone-related genes seems to persist across cancers.

Detection of Plasma EGFR Mutations in NSCLC Patients with a Validated ddPCR Lung cfDNA Assay.

Purpose: The clinical utility of cell-free DNA (cfDNA) to assess EGFR mutations is increasing. However, there are limited studies determining their clinical validity and utility. The value of cfDNA assays in cancer management remains controversial. Methods: In this study, we first evaluated the analytical performance of the ddPCR Lung cfDNA Assay. We next analyzed the concordance of the results with tissue amplification refractory mutation system PCR (ARMS-PCR) and plasma next-generation sequencing (NGS) genotyping. Finally, we assessed its clinical utility by exploring the association of cfDNA EGFR mutations with metastatic sites and the efficacy of EGFR-TKIs treatment. Results: The ddPCR Lung cfDNA Assay demonstrated a limit of blank of 1 droplet per reaction, an analytical specificity of 100%, and detection limit of 0.05%, 0.05%, and 0.1% for E746_A750del, L858R, and T790M, respectively. With tissue ARMS-PCR as a standard for comparison, the clinical sensitivity and specificity of ddPCR were 62.5% (15/24) and 100% (82/82) for E746_A750del, and 75.0% (15/20) and 94.2% (81/86) for L858R, respectively. The ddPCR showed high concordance with NGS in determining cfDNA EGFR mutations. Patients with bone and/or brain metastasis showed a higher detection rate and mutant abundance of cfDNA EGFR mutations compared to those with other sites of metastasis. Moreover, EGFR-TKIs treatment was effective in patients with sensitive EGFR mutations in either plasma cfDNA or tumor tissue-derived DNA. Conclusions: We validated in this study that the ddPCR Lung cfDNA Assay is reliable for detection of EGFR mutations in lung cancers, in terms of analytical performance, clinical validity and utility.

Absolute quantification and analysis of extracellular vesicle lncRNAs from the peripheral blood of patients with lung cancer based on multi-colour fluorescence chip-based digital PCR.

Emerging evidence indicates that extracellular vesicle (EV) long non-coding ribonucleic acids (lncRNAs) in lung cancer may be clinically useful biomarkers for early diagnosis using liquid biopsy. However, the extremely low quantities of EV-lncRNAs in peripheral blood are a major challenge for multi-target detection. In this study, we developed a new multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis chip, and then demonstrated its ability to quickly and accurately analyse the levels of two target genes and one reference gene from peripheral blood. Under the miDER assay, the limit of detection of the target gene from peripheral blood was 10 copies/µL. Based on multiplex assay, the expression levels of two lung cancer-related genes (SLC9A3-AS1 and PCAT6) in patients with lung cancer (n = 32) and healthy controls (n = 30) showed a significant difference between the two groups (P < 0.001; two-tailed t-test). A receiver operating characteristic (ROC) curve analysis was used to evaluate the discrimination ability of these lncRNAs. The combination of two lncRNAs in the miDER assay yielded a higher area under curve (AUC) value of 0.811 (95% CI = 0.705-0.918). Moreover, to determine the absolute quantitation capacity of the miDER assay, we compared the results to those obtained by quantitative real-time polymerase chain reaction (qPCR), demonstrating that the miDER assay is more sensitive than qPCR. The multiplex assay based on the miDER could provide a new solution for the multi-index combined detection of trace EV-lncRNAs in body fluids and demonstrate the use of EV-lncRNAs as biomarkers for lung tumour biopsy.

Multiplexed Detection of Mutant Circulating Tumor DNA Using Peptide Nucleic Acid Clamping Asymmetric Polymerase Chain Reaction and Liquidchip.

Circulating tumor DNA (ctDNA) in blood has been investigated as a feasible substitute for genetic alterations in tumor tissues to predict and assess drug responses, but current techniques of screening clinical relevant mutations still have great limitations in sensitivity, specificity, or multiplexed detection because of highly fragmented ctDNA and its low concentration in a high background of normal DNA. In this study, we developed PNA-aPCR-Liquidchip (PAPL), a novel method that aims to detect multiple mutant ctDNA. In order to demonstrate its utility, we analyzed three high frequent epidermal growth factor receptor (EGFR) mutations (exon 19 deletion, L858R, and T790M) in non-small cell lung cancer (NSCLC). Multiplexed analyses indicated that this method has high specificity and sensitivity which could detect down to 2∼5 copies of mutant EGFR in a background of 10,000 copies of wild-type genomic DNA, achieving the mutant abundance of 0.02%∼0.05%. Furthermore, PAPL had no significant differences with droplet digital PCR (ddPCR) in plasma cell-free DNA (cfDNA) detection. Thus, PAPL can be used to detect EGFR mutations in NSCLC patients' plasma, indicating that this method has great potential for application in the context of precision medicine based on mutant ctDNA detection.

Value of folate receptor-positive circulating tumour cells in the clinical management of indeterminate lung nodules: A non-invasive biomarker for predicting malignancy and tumour invasiveness.

BACKGROUND: Non-invasive lung adenocarcinoma could benefit from limited resection, nonetheless, there is a lack of method to determine preoperative tumour invasiveness. We aimed to investigate whether folate receptor-positive circulating tumour cells (FR+-CTCs) in combination with maximum tumour diameter (MTD) determines, before surgery, the invasiveness of small-sized, indeterminate solitary pulmonary nodules (SPNs). METHODS: A total of 382 patients with suspicious lung adenocarcinoma on computed tomography who were expected to undergo lung resection were enrolled in this study at three participating institutes and randomly assigned into training and validation cohorts. Before surgery, 3 mL peripheral blood was collected from all participants. FR+-CTCs were analyzed using immunomagnetic leukocyte depletion and quantitated by ligand-targeted PCR method. After surgery, the resected tissues were diagnosed by pathologists according to IASLC/ATS/ERS classification. FINDINGS: FR+-CTC levels in the peripheral blood can differentiate benign from malignant nodules with a sensitivity of 78·6%-82·7% and a specificity of 68·8%-78·4%. Both FR+-CTC and MTD are independent predictive indices of invasive tumours for lung adenocarcinoma ≤2 cm based on multivariate analyses. Further, FR+-CTC count in combination with MTD can differentiate non-invasive cancers from invasive cancers with a sensitivity of 63·6%-81·8% and a specificity of 71·4%-89·7%. INTERPRETATION: Detection of FR+-CTC is a reliable method to differentiate malignancy of indeterminate SPNs. Combining of FR+-CTC count and MTD could possibly enhance preoperative determination of the invasiveness of lung nodules and guide surgeons to select limited lung resection and avoid overtreatment for patients with non-invasive lesions. FUND: None.

Heterogeneous mutation pattern in tumor tissue and circulating tumor DNA warrants parallel NGS panel testing.

Liquid biopsy by genotyping circulating tumor DNA (ctDNA) has provided a non-invasive approach in assessing tumor genomic alterations in clinical oncology. However, emerging evidence in clinical settings has shown significant discordance in the genomic alterations between matched tumor tissue and blood ctDNA samples, and even between the same set of blood samples analyzed on different testing platforms. Thus, it is necessary to study underlying causes of discrepancies in these studies by genotyping tumor tissue and ctDNA in parallel using next generation sequencing (NGS) panels based on the same technology. Here we enrolled 56 non-small-cell lung cancer (NSCLC) patients and evaluated tumor tissue genotyping and ctDNA based liquid biopsy by parallel NGS panel testing and compared different sample preparation conditions. Somatic mutations in plasma cell-free DNA (cfDNA) were detected in 63.6% patients with early-stage NSCLC and 60% patients with advanced-stage NSCLC. The overall concordance between matched formalin-fixed paraffin-embedded sample and cfDNA was 54.6% in early-stage NSCLC patients and 80% in advanced-stage NSCLC patients. The positive concordance rate was 44.4% and 71.4% in early-stage and advanced-stage patients, respectively. Using fresh frozen tumor samples did not improve the overall concordance rate between matched tumor tissue and cfDNA. Processing blood samples beyond 4 h after blood draw significantly decreased the detection rate of somatic mutations in cfDNA. Thus, the concordance rate between tumor tissue-based and ctDNA-based genotyping in clinical samples can be affected by multiple pre-analytical, analytical and biologic factors. Parallel NGS panel testing on both sample types for each patient may be warranted for effective guidance of cancer targeted therapies and possible early detection of cancer.