Mutation Spectrum of EGFR From 21,324 Chinese Patients With Non-Small Cell Lung Cancer (NSCLC) Successfully Tested by Multiple Methods in a CAP-Accredited Laboratory.

Genotyping epidermal growth factor receptor (EGFR) gene in patients with advanced non-small cell lung cancers (NSCLC) is essential for identifying those patients who may benefit from targeted therapies. Systemically evaluating EGFR mutation detection rates of different methods currently used in clinical setting will provide valuable information to clinicians and laboratory scientists who take care of NSCLC patients. This study retrospectively reviewed the EGFR data obtained in our laboratory in last 10 years. A total of 21,324 NSCLC cases successfully underwent EGFR genotyping for clinical therapeutic purpose, including 5,244 cases tested by Sanger sequencing, 13,329 cases tested by real-time PCR, and 2,751 tested by next-generation sequencing (NGS). The average EGFR mutation rate was 45.1%, with 40.3% identified by Sanger sequencing, 46.5% by real-time PCR and 47.5% by NGS. Of these cases with EGFR mutations identified, 93.3% of them harbored a single EGFR mutation (92.1% with 19del or L858R, and 7.9% with uncommon mutations) and 6.7% harbored complex EGFR mutations. Of the 72 distinct EGFR variants identified in this study, 15 of them (single or complex EGFR mutations) were newly identified in NSCLC. For these cases with EGFR mutations tested by NGS, 65.3% of them also carried tumor-related variants in some non-EGFR genes and about one third of them were considered candidates of targeted drugs. NGS method showed advantages over Sanger sequencing and real-time PCR not only by providing the highest mutation detection rate of EGFR but also by identifying actionable non-EGFR mutations with targeted drugs in clinical setting.

Parallel Analyses of Somatic Mutations in Plasma Circulating Tumor DNA (ctDNA) and Matched Tumor Tissues in Early-Stage Breast Cancer.

PURPOSE: Early detection and intervention can decrease the mortality of breast cancer significantly. Assessments of genetic/genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of early-stage breast cancer.Experimental Design: In this study, 861 serial plasma and matched tissue specimens from 102 patients with early-stage breast cancer who need chemotherapy and 50 individuals with benign breast tumors were deeply sequenced via next-generation sequencing (NGS) techniques using large gene panels. RESULTS: Cancer tissues in this cohort of patients showed profound intratumor heterogeneities (ITHGs) that were properly reflected by ctDNA testing. Integrating the ctDNA detection rate of 74.2% in this cohort with the corresponding predictive results based on Breast Imaging Reporting and Data System classification (BI-RADS) could increase the positive predictive value up to 92% and potentially dramatically reduce surgical overtreatment. Patients with positive ctDNA after surgery showed a higher percentage of lymph node metastasis, indicating potential recurrence and remote metastasis. The ctDNA-positive rates were significantly decreased after chemotherapy in basal-like and Her2+ tumor subtypes, but were persistent despite chemotherapy in luminal type. The tumor mutation burden in blood (bTMB) assessed on the basis of ctDNA testing was positively correlated with the TMB in tumor tissues (tTMB), providing a candidate biomarker warranting further study of its potentials used for precise immunotherapy in cancer. CONCLUSIONS: These data showed that ctDNA evaluation is a feasible, sensitive, and specific biomarker for diagnosis and differential diagnosis of patients with early-stage breast cancer who need chemotherapy.

Loop Replacement Enhances the Ancestral Antibacterial Function of a Bifunctional Scorpion Toxin.

On the basis of the evolutionary relationship between scorpion toxins targeting K⁺ channels (KTxs) and antibacterial defensins (Zhu S., Peigneur S., Gao B., Umetsu Y., Ohki S., Tytgat J. Experimental conversion of a defensin into a neurotoxin: Implications for origin of toxic function. Mol. Biol. Evol. 2014, 31, 546⁻559), we performed protein engineering experiments to modify a bifunctional KTx (i.e., weak inhibitory activities on both K⁺ channels and bacteria) via substituting its carboxyl loop with the structurally equivalent loop of contemporary defensins. As expected, the engineered peptide (named MeuTXKα3-KFGGI) remarkably improved the antibacterial activity, particularly on some Gram-positive bacteria, including several antibiotic-resistant opportunistic pathogens. Compared with the unmodified toxin, its antibacterial spectrum also enlarged. Our work provides a new method to enhance the antibacterial activity of bifunctional scorpion venom peptides, which might be useful in engineering other proteins with an ancestral activity.

Evaluating the potential of a loop-extended scorpion toxin-like peptide as a protein scaffold.

Grafting of exogenous bioactive sites or functional motifs onto structurally stable scaffolds to gain new functions represents an important research direction in protein engineering. Some engineered proteins have been developed into therapeutic drugs. MeuNaTxα-3 (abbreviated as MT-3) is a newly characterized scorpion sodium channel toxin-like peptide isolated from the venom of the scorpion Mesobuthus eupeus, which contains a rigid scaffold highly similar to classical scorpion sodium channel toxins and an extension of eight amino acids in its J-loop region. This extended loop constitutes a flexible region extruded from the scaffold and could be substituted by exogenous functional sequences. In this study, we experimentally evaluated the scaffold potential of MT-3 through grafting two small antimicrobial motifs to replace residues within the loop. Functional assays showed that the two engineered molecules exhibited elevated antimicrobial potency, as compared with the unmodified scaffold, without structural disruption, providing experimental evidence in favor of MT-3 as a promising scaffold in protein engineering.

Target-Driven Positive Selection at Hot Spots of Scorpion Toxins Uncovers Their Potential in Design of Insecticides.

Positive selection sites (PSSs), a class of amino acid sites with an excess of nonsynonymous to synonymous substitutions, are indicators of adaptive molecular evolution and have been detected in many protein families involved in a diversity of biological processes by statistical approaches. However, few studies are conducted to evaluate their functional significance and the driving force behind the evolution (i.e., agent of selection). Scorpion α-toxins are a class of multigene family of peptide neurotoxins affecting voltage-gated Na(+ )(Nav) channels, whose members exhibit differential potency and preference for insect and mammalian Nav channels. In this study, we undertook a systematical molecular dissection of nearly all the PSSs newly characterized in the Mesobuthus α-toxin family and a two-residue insertion ((19)AlaPhe(20)) located within a positively selected loop via mutational analysis of α-like MeuNaTxα-5, one member affecting both insect and mammalian Nav channels. This allows to identify hot-spot residues on its functional face involved in interaction with the receptor site of Nav channels, which comprises two PSSs (Ile(40) and Leu(41)) and the small insertion, both located on two spatially separated functional loops. Mutations at these hot-spots resulted in a remarkably decreased anti-mammalian activity in MeuNaTxα-5 with partially impaired or enhanced insecticide activity, suggesting the potential of PSSs in designing promising candidate insecticides from scorpion α-like toxins. Based on an experiment-guided toxin-channel complex model and high evolutionary variability in the receptor site of predators and prey of scorpions, we provide new evidence for target-driven adaptive evolution of scorpion toxins to deal with their targets' diversity.