Lung cancer still contributes to nearly one‐quarter cancer‐related deaths in the past decades, despite the rapid development of targeted therapy and immunotherapy in non‐small cell lung cancer (NSCLC). The development and availability of comprehensive genomic profiling make the classification of NSCLC more precise and personalized. Most treatment decisions of advanced‐stage NSCLC have been made based on the genetic features and PD‐L1 expression of patients. For the past 2 years, more than 10 therapeutic strategies have been approved as first‐line treatment for certain subgroups of NSCLC. However, some major challenges remain, including drug resistance and low rate of overall survival. Therefore, we discuss and review the therapeutic strategies of NSCLC, and focus on the development of targeted therapy and immunotherapy in advanced‐stage NSCLC. Based on the latest guidelines, we provide an updated summary on the standard treatment for NSCLC. At last, we discussed several potential therapies for NSCLC. The development of new drugs and combination therapies both provide promising therapeutic effects on NSCLC. Based on the development of targeted therapy and immunotherapy, the treatment strategies for NSCLC have been profoundly changed. After evaluation of the incidental finding of nodule, patients are managed to routine follow‐up or diagnosis of NSCLC. Patients with NSCLC should receive molecular testing and PD‐L1 testing to make final treatment decision. For resectable NSCLC with targetable mutations, targeted therapy as adjuvant therapy shows promising effects. Immunotherapy has also been investigated as both neoadjuvant and adjuvant therapy. For unresectable advanced NSCLC with driver gene positive, targeted therapy and immunotherapy play an essential role in anticancer treatment. This review comprehensively presents the current landscape of targeted therapy and immunotherapy in NSCLC. An additional 2.7% deaths are due to second‐hand smoke.Tobacco smoking is directly associated with NSCLC and there are more than 50 carcinogens in the tobacco smoke.EGFR  mutation, rearrangements in ALK (anaplastic lymphoma kinase), fusions in ROS1(ROS proto‐oncogene 1), BRAF  V600E, NTRK (neurotrophic tyrosine receptor kinase)1/2/3 gene fusion, MET  exon 14 skipping, and RET (rearranged during transfection) rearrangement, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of driven gene mutation‐positive NSCLC patients. Because of comprehensive genomic profiling, other genetic aberrances in NSCLC, such as mutations in Kirsten rat sarcoma (KRAS), amplification of human epidermal growth factor receptor‐2 (HER2), and other genotypes of the driver genes, have been thought highly targetable and investigated in preclinical and clinical trials.EGFR mutation, ALK rearrangements, or ROS1 fusions, but usually gets alterations in RTKs, CDKN2A, PTEN, MLL2, HLA‐A, NOTCH1, and RB1.Surgery is recommended for early‐stage (stage I–II) NSCLC patients, whereas more than 70% of NSCLC are diagnosed as advanced stage (stage III–IV).Despite the development of the therapeutic strategies and improved survival for NSCLC, some major concerns remain challenging, such as the resistance to targeted therapy and immunotherapy, optimal combinations of the current treatment regimens, and investigation for new potential targets. Besides, with rapid development of targeted therapy during recent years, several breakthrough TKIs have been approved clinically. Based on the clinical management of NSCLC, we provide an overview for the treatment of advanced NSCLC and focus on targeted therapy and immunotherapy. Meanwhile, the important completed and ongoing clinical trials of both targeted therapy and immunotherapy are summarized in this review.2With the introduction of surgery and cytotoxic chemotherapy, the prognosis of NSCLC patients was improved for the first time. Through the development of molecular biology, new therapies, such as antiangiogenesis therapy, targeted therapy, and immunotherapy, have yielded encouraging therapeutic effects in advanced NSCLC. Remarkable changes have been made in the treatment of NSCLC during last decades Figure .2.1EGFR, ALK, and ROS1 are initially recommended as three targetable oncogenic drivers, which must be tested if the tissue is limited for next‐generation sequencing (NGS) panel.EGFR, ALK, KRAS, ROS1, BRAF, NTRK1/2/3, MET, RET, and PD‐L1 expression. DNA sequencing is a traditional way to identify genomic mutations, which requires relatively enriched tumor cells. Mutation‐specific polymerase chain reaction (PCR) kits have been used in clinical laboratory for its high sensitivity with only 1–5% tumor cells.ALK, ROS1, RET, and NTRK.EGFR.ALK D5F3 CDx Assay is the only IHC test approved by the U.S. FDA for ALK inhibitors.NSCLC is a molecularly heterogeneous disease, which makes early identification of tumor genotype critical. Companion diagnostic assays are usually released by the U.S. FDA for approval of targeted agents, in order to identify essential genomic alterations before initiating therapy.2.2EGFR mutation‐positive NSCLC, targeted therapy as adjuvant treatment, has improved the survival of NSCLC patients. For instance, the 24‐month DFS is 89% in osimertinib group versus 52% in the placebo group, with reduced local relapse and metastasis.EGFR or ALK mutation (EA5142), and adjuvant EGFR (A081105) or ALK (E4512) TKIs for NSCLC with EGFR or ALK mutation.The treatment strategy for early‐stage NSCLC is based on surgery Figure . Surgery2.3NSCLC patients in advanced phases usually lose the chance for surgery. For advanced NSCLC, combination chemotherapy of 4–6 cycles followed by observation is used to be treated as standard care.3Patients with advanced NSCLC benefit a lot from the development of targeted therapy. It is estimated that more than 65% of patients with advanced NSCLC have a potentially targetable genomic alteration.3.1EGFR gene is located on the short arm of chromosome 7 (7p11.2) and contains 28 exons and 27 introns. Mutations clustering around the ATP‐binding pocket of the tyrosine kinase domain lead to constituent, ligand‐independent activation of EGFR, which results in inappropriate activation of the antiapoptotic Ras signaling pathway and abnormal proliferation of cancer cells.EGFR mutations in NSCLC occur in approximately 40% of Asian patients, about 10% of non‐Asian patients, and most of them are LUAD, young females, and nonsmoker.EGFR in NSCLC (approximately 85%) include exon 19 deletions and a point mutation on exon 21 , known as sensitizing mutations, which are responsible for oral TKIs targeting EGFR. Other mutations like exon 20 insertions and point mutations on exon 18 are less common.EGFR exon 20 are a heterogeneous group, some of which are predictions for the response to EGFR TKIs, such as T790M mutation.EGFR. Results from recent data showed that patients without sensitizing EGFR mutation should not receive EGFR TKIs in any line of therapy.There are 58 receptor tyrosine kinases (RTKs) in humans, and the epidermal growth factor receptor  is one of the first RTKs regarded as an anticancer target and brings significant improvement in the survival of NSCLC patients.EGFR mutation.EGFR mutations  in terms of progression‐free survival (PFS), objective response rate (ORR), and quality of life, whereas these EGFR TKIs showed no advantage in patients without such mutation.EGFR sensitizing mutation‐positive advanced NSCLC patients in 2009 and 2013, respectively. There is no significant difference between the two EGFR TKIs in the therapeutic effects based on the results of randomized phase III trials.EGFR mutation. The results of ICOGEN trial in China, a double‐blind, head‐to‐head phase III study containing 399 patients, showed that the median PFS of icotinib arm was 137 days as compared to that of gefitinib arm was 102 days.EGFR mutation. This approval was based on the results from EVIDENCE trial, in which icotinib showed an improved median DFS of 46.9 months compared with 22.1 months in standard chemotherapy group.EGFR mutation‐positive NSCLC patients.Three generations of EGFR TKIs have been used in clinical applications. First‐generation EGFR TKIs, including gefitinib, erlotinib, and icotinib, reversibly bind to EGFR and competitively inhibit the binding of ATP to tyrosine kinase domain. Gefitinib and erlotinib were first approved by the U.S. FDA for treatment of patients with advanced NSCLC (unselected), who have failed in standard chemotherapy.EGFR mutations compared with platinum‐based chemotherapy.EGFR exon 19 deletions or exon 21 (L858R) substitution mutations and got a broadened indication as the first‐line treatment of advanced NSCLC patients with nonresistant EGFR mutations. Dacomitinib was approved by the U.S. FDA in 2018 as first‐line treatment for advanced NSCLC patients with EGFR exon 19 deletion or exon 21 L858R substitution mutations. The approval was based on a randomized, multicenter, and open‐label clinical trial (ARCHER 1050), in which the median PFS was 14.7 and 9.2 months in the dacomitinib and gefitinib arms, respectively.Afatinib and dacomitinib, two irreversible ERBB‐family (pan‐HER) inhibitors, are the second‐generation EGFR TKIs. Unlike the first‐generation, they bind to ATP‐binding domain of EGFR irreversibly and less selectively. Based on the results of three clinical trials, LUX‐Lung 2, LUX‐Lung 3, and LUX‐Lung 6, afatinib showed better ORR (approximately 70%) and prolonged the PFS (approximately 1 year) in advanced NSCLC patients with EGFR sensitizing mutations and T790M mutation. T790M is the most common second mutation causing drug resistance, which is referred to a threonine‐tomethionine substitution on codon 790 in exon 20 (T790M).EGFR T790M mutation‐positive patients with disease progression on or after EGFR TKI therapy. Approval went through an accelerated process based on the promising results of AURA extension and AURA 2.EGFR mutations or as an adjuvant therapy when surgery patients with sensitizing EGFR mutations.EGFR T790M, who got disease progression on or after EGFR TKI therapy. This approval was based on the findings of an open‐label phase II study, APOLLO, in which the median PFS and ORR of patients with progressed NSCLC harboring EGFR T790M mutation treated with aulmonertinib was 12.3 months and 68.9%. Of note, aulmonertinib induced an ORR of 61.5% in patients with CNS metastasis.EGFR mutations. Aumolertinib significantly improved PFS to 19.3 months compared to 9.9 months in gefitinb arm. As for side effects, the incidence of rash or diarrhea was markedly decreased in aumolertinib arm.EGFR T790M mutation‐positive advanced NSCLC patients treated with furmonertinib showed an ORR of 74.1% and PFS of 9.6 months.EGFR mutations.Nearly, all patients received first‐ or second‐generation EGFR TKIs treatment eventually acquired drug resistance, leading to disease progression.Targeted therapy strategy contains two main approaches: mAbs and small‐molecule inhibitors (SMIs). The advantages and disadvantages between mAbs and SMI have been well discussed in Ref. EGFR mutations, exon 19 deletions (Del19) and the L858R point mutation, which account for about 85% of observed EGFR mutations in NSCLC.EGFR mutations have been accessed via post‐hoc analyses of clinical trials and discussed in Ref. At present, EGFR TKIs have been approved for the treatment of sensitizing EGFR exon 20 insertion, which is associated with poor prognosis.EGFR exon 20 insertion, amivantamab‐vmjw and mobocertinib, have won the U.S. FDA approval as second‐line treatment for EGFR exon 20 insertion‐positive advanced NSCLC patients. Amivantamab‐vmjw, a bispecific mAb targeting EGFR and MET, has shown an ORR of 40% with a median response duration of 11.1 months.During the last 2 years, breakthroughs have been made in targeted therapy for 3.2ALK as a fusion gene partner was found in anaplastic large‐cell lymphoma in 1994.ALK rearrangements in NSCLC have been found in 2007 by the initial discovery of a fusion gene containing parts of the echinoderm microtubule‐associated protein‐like 4 (EML4) gene and ALK gene.ELM4‐ALK fusion identified and EML4‐ALK, variant 1 is the most common and well‐studied one.EML4 and ALK genes are both located on chromosome 2p, and the variants of EML4‐ALK fusion depend on the fusion breakpoint in the EML4 gene, with the breakpoint in ALK is usually at exon 20.ALK are more than ELM4, others like huntingtin‐interacting protein 1 (HIP1),kinesin family member 5B (KIF5B),kinesin light chain 1 (KLC1),translocated promoter region (TPR),ALK arrangement‐driven tumors account for about 5% of NSCLC, and most of them are Asian, men, and never/light smokers and are likely to be adenocarcinomas.EGFR mutations, such as exon 19 deletion is associated with better prognosis, it is not clear whether specific genetic alteration in ALK mutation is associated with therapeutic response.ALK is an RTK naturally expressed in human tissues, such as brain, small intestine, and testis, without complete understanding of its function, but shows strong oncogenic effects.ALK arrangements.ALK rearrangements.Five ALK TKIs, including crizotinib, ceritinib, alectinib, brigatinib, and loralatinib, have been approved as targeted therapy for advanced NSCLC patients who are confirmed to have ALK mutations, G1202R and F1174C.ALK‐positive advanced NSCLC in May 2017. Alectinib, another second‐generation ALK TKI, has shown an ORR of approximately 50% of patients failed in crizotinib therapy, with median PFS of more than 8 months. A phase III clinical trial (ALEX) revealed that patients treated with alectinib showed prolonged PFS of 26 months compared with 10 months in crizotinib group. The ORR was 79% for alectinib and 72% for crizotinib.Ceritinib (LDK378) and alectinib are two second‐generation ALK TKIs approved by the U.S. FDA for NSCLC patients failed in or tolerate on crizotinib therapy.ALK‐positive NSCLC patients.ALK‐positive NSCLC patients were enrolled in this trial. The median PFS and ORR for patients treated with brigatinib was 24 months and 74%, respectively, compared with 11.1 months and 62% for those treated with crizotinib, respectively.Brigatinib is a small molecular inhibitor targeting both EGFR and ALK, which has been confirmed to overcome the osimertinib‐resistant C797S mutation and expected to be the next‐generation EGFR TKI.ALK‐positive NSCLC patients.ALK and ROS1‐resistance mutation, such as I1171T and G1202R.ALK mutations. Lorlatinib has shown obvious advantages, including penetration into CNS, fewer side effects, and less drug resistance, compared with former‐generations ALK TKIs.Recently, in March 2021, a former second‐line ALK TKI, lorlatinib, has been approved as a first‐line treatment for advanced 3.3ROS1 gene on chromosome 6q22. The biologic function of wild‐type ROS1 is not well identified and there has no specific ligand of ROS1 been found.CD74 (most common),FIG ,SLC34A2 (so lute carrier family 34 member 2),ROS1 fusion partner genes according to Catalogue of Somatic Mutations in Cancer (COSMIC) data bese, and ROS1 arrangements have been observed in 1–2% NSCLC, most of which are adenocarcinomas, female, and never or light smokers.ROS1 fusion detection is FISH assay.ROS1 is an oncogenic RTK of insulin receptor family encoded by the ROS1 amide acid sequence shares 49% homology with ALK in the kinase domain and 77% homology at the ATP‐binding area.ALK‐positive NSCLC, is one of the two targeted agents approved by the U.S. FDA in 2016 for ROS1‐positive advanced NSCLC. This approval was based on a single‐arm study in 50 advanced NSCLC patients with ROS1 arrangements. The ORR was approximately 66% and the PFS was 18.3 months.ROS1‐positive NSCLC and neurotrophic tyrosine receptor kinase (NTRK) gene fusion‐positive solid tumor, including NSCLC in August 2019. The ORR of advanced NSCLC patients treated by entrectinib was 78% and the response duration was 24.6 months, with ability to penetrate blood–brain barrier.The 3.4BRAF mutations have been observed in 3–8% of NSCLC, most of which are adenocarcinomas and smokers.BRAF mutations  is a single‐point mutation at residue 600 of exon 15, where valine is replaced by glutamate .BRAF gene encodes a threonine/serine protein kinase, which is involved in MAPK/ERK signaling pathway. V600E mutation leads to uncontrolled activation of BRAF, resulting in abnormal cell proliferation.BRAF mutations can induce either activation or inactivation of BRAF.BRAF V600E mutation‐positive advanced NSCLC has been approved by the U.S. FDA in 2017. The therapeutic effects of dabrafenib (BRAF TKI) and trametinib (MEK TKI) combination were confirmed by a nonrandomized, noncomparative, and open‐label trial. The ORRs were 63% and 61% in previously treated patients and treatment‐naive patients, respectively. However, the ORR for patients who received just single agent of dabrafenib was 27%.One targeted therapy for 3.5KRAS is an oncogene which belongs to RAS GTPase family and in control of crucial cellular pathway, including RAF/MEK/ERKandPI3K/AKT.KRAS mutations are common in NSCLC, accounting for 25% of adenocarcinomas, especially in ever/heavy smokers of western countries.KRAS lead to continuous activation of KRAS and uncontrolled cell proliferation. However, the targeted therapy for KRAS is limited and patients with KRAS mutations have poor prognosis with a median survival of 2.4 years.KRAS alteration, G12C (a point mutation that guanine is replaced by cysteine).KRAS alterations have been well reviewed in Ref. KRAS‐positive NSCLC patients in combination with chemotherapy.KRAS G12C‐mutated NSCLC in May 2021. The approval for sotorasib (Lumakras) was based on the results of a single‐arm and open‐label phase I/II trial, CodeBreaK 100. KRAS G12C‐positive advanced NSCLC patients whose disease had progressed on or after chemotherapy were enrolled. The ORR was 36% with a median duration of 10 months.3.6RET encodes a tyrosine kinase receptor on cell surface, which is involved in several crucial signaling pathways, including MAPK, PI3K, JAK/STAT, PKA, and PKC pathways.RET arrangements (RET fusion) have been observed in 1–2% NSCLC, mostly adenocarcinomas, never/light smokers, and younger patients.RET have been identified, among which the intron 15 of KIF5B is the most common and widely studied for targeted therapy.RET‐positive NSCLC patients showed that the response rates  to cabozantinib, vandetanib, and sunitinib were 37%, 18%, and 22%, respectively. Further responses were observed with lenvantinib and nintedanib‐treated patients.RET‐positive NSCLC, and several RET‐specific agents have entered clinical trials. Selpercatinib (LOXO292), pralsetinib (BLU‐667), BOS172738,RET‐positive advanced NSCLC.3.7MET  gene encodes hepatocyte growth factor receptor, the phosphorylation of which leads to the activation of several cellular signaling pathways, including MAPK, PI3K, STAT, and so on.MET amplification is usually associated with EGFR TKIs resistance. MET exon 14 skipping is the most common pattern of MET mutations, which leads to increased stability and continuous activation of MET protein.EGFR‐ or KRAS‐positive NSCLC patients.MET exon 14 skipping‐positive patients is much poor than driver gene‐negative patients with a median OS of 6.7 versus 11.2 months, which makes MET targeted therapy important.MET exon 14 skipping. Crizotinib, as an MET, ALK, and ROS1 multitargeted inhibitor, was approved for the second‐line treatment of MET exon 14 skipping‐positive NSCLC patients.MET exon 14 skipping mutation‐positive advanced NSCLC. The efficacy of capmatinib was demonstrated in a phase II trial enrolling patients with confirmed MET exon 14 skipping. Previously untreated patients benefited more from capmatinib treatment with an ORR of 68% and a median duration of 12.6 months, compared with 41% and 9.7 months in previously treated patients.MET exon 14 skipping mutation. The ORR of savolitinib‐treated group was 42.9%, with a median PFS of 6.8 months and a median OS of 12.5 months.The 3.8NTRK (neurotrophic tropomyosin‐related kinases) genes 1, 2, and 3 encode tropomyosin receptor kinases (TRKs) A, B, and C, respectively. NTRK fusion has been observed in 1–2% of NSCLC patients and assumed as targetable mutations.NTRK gene fusion‐positive solid tumor (including NSCLC), who have progressed on their primary treatment or have no satisfactory standard therapy.3.9HER2 amplification and overexpression have been observed in approximately 30% and 10% of lung cancer, respectively.HER2 driving mutations, which is rare and present in about 2–3% of patients, mostly women, never/light smoker, and adenocarcinoma.EGFR mutation, the common mutations in HER2 occur in exon 20 by insertion of DNA bases.HER2 (ERBB2), with EGFR, HER3, and HER4, belongs to ERBB RTK family. 3.10Besides targeting genomic alterations, inhibition tumor vascular formation by targeting angiogenic factors is also a promising and classic anticancer strategy. Vascular supply is essential for the growth and progression of solid tumor, without which tumors remain stable and localized.EGFR mutations (19.4 vs. 12.4 months) compared with erlotinib single‐agent group.Bevacizumab  and ramucirumab  are two mAbs approved by the U.S. FDA for the treatment of NSCLC. Bevacizumab, which is the first antiangiogenic drug, inhibits angiogenesis through binding and neutralizing all VEGF isoforms.TKIs targeting VEGF/VEGFR, platelet‐derived growth factor/receptor (PDGF/PDGFR), fibroblast growth factor/receptor (FGF/FGFR), and c‐Kit demonstrate effects on inhibition angiogenesis, including sorafenib, sunitinib, vandetanib, nintedanib, and anlotinib.The angiogenesis process is also negatively regulated by endostatin, which is thought to be a potential target in antiangiogenic strategy.4Since the first TKI, gefitinib, has been introduced to treat NSCLC in the late 1990s, the development of the therapeutic strategies for NSCLC progressed rapidly. Despite the promising effects of TKIs, unavoidable drug resistance has been observed in most patients. The increased heterogeneity within the tumor during targeted therapy is associated with poor therapeutic effects.4.1EGFR‐sensitizing mutations in exon 19 or 21.EGFR. However, resistance to osimertinib has also been observed in clinic. The most common secondary mutation related to osimertinib is EGFR‐C797S,EGFR also shows resistance to osimertinib.Almost all patients have acquired resistance after first‐ or second‐generation EGFR TKIs treatment. The median duration time is less than 1 year.BRAF (G469A or V600E) in resistance to early or third generation of EGFR TKIs usually results in the reactivation of MAPK pathway.KRAS mutation, including mutations in PIKC3A and loss of PTEN, is a negative predictor of EGFR TKIs treatment.Alternative pathway activation is common during the process of drug resistance of EGFR TKIs, including MAPK, PI3K/AKT, JAK‐STAT3, and SRC pathways. For example, acquisition of mutations of 4.2ALK mutations are relatively variable in patients with drug resistance.ALK have shown resistance to crizotinib via physically interfering TKI binding.ALK amplification.ALK‐G1202R mutation occurs in less than 2% of patients with resistance to ALK TKIs and is assumed to show resistance to almost all approved ALK TKIs.ALK‐G1202R mutation with an ORR of 44% in a phase III clinical trial.ALK mutations, including I1171T and V1180.The secondary KRAS amplification or MEK mutation in patients with resistance to ALK TKIs was observed.Activation of MAPK pathway via 4.3ROS1 mutations are structural analogues to ALK mutations.ROS1 (L2026M).ROS1‐G2032R and ROS1‐D2033N are structural analogous of ALK‐G1202R and ALK‐D1203N mutations, respectively, which show resistance to crizotinib. In a small sample study, ROS1‐G2032R is the most common mutation showing resistance to crizotinib.ROS1 and ALK share similar structure of tyrosine kinase domains, therefore, many 4.4RET is a gatekeeper mutation and responsible for the resistance to cabozantinib. It is assumed that ponatinib is the most potent RET TKI to overcome the RET TKIs‐associated drug resistance.HER2‐C805S, has been reported at resistance to HER2 TKI therapy in HER2‐mutated NSCLC,PIK3CA has been reported in NSCLC patients with HER2 TKIs resistance, and combined therapy of mTOR inhibitor showed therapeutic response.During the treatment of TKIs of RET, HER2, and MET for NSCLC patients, the medium duration is usually less than 12 months. V804L mutation of 4.5MET amplification occurs with 5–20% incidence and leads to resistance to early generation of EGFR TKIs.MET amplification and resistance to prior EGFR TKI treatment, MET inhibitor capmatinib combined with gefitinib showed the response rate of about 15%.EML4‐ALK rearrangement is associated with resistance to EGFR TKIs.Secondary mutations other than targeted gene (off‐target resistance) are also common in the process of drug resistance. During the treatment of targeted therapy, alterations of genetic characteristics are usually associated with drug resistance. In NSCLC patients who have progressed on EGFR TKIs, Besides those targetable mutations in NSCLC, some proteins also show abnormal expression during drug resistance. Increased expression of AXL receptor tyrosine kinase (AXL) has been observed in the samples of NSCLC patients resistant to EGFR, ALK, or RET TKIs treatment, indicating that AXL TKIs might be an alternative for combination therapy in targeted therapy for NSCLC.4.6In a group of NSCLC patients with resistance to EGFR or ALK TKIs, histological transformation from an NSCLC to a small‐cell lung cancer histology has been observed.5The emergency of targeted therapy has improved the survival of certain groups of NSCLC patients; however, the 5‐year survival is still not satisfying.5.1The expression of PD‐L1 is used to be treated as a biomarker to assess the response of patients to immunotherapy.5.2The PD‐1 molecule is mainly expressed on T/B cells, NK, and MDSCs. PD‐L1 and PD‐L2 are two ligands induced by inflammatory signals. Their interactions downregulate the activation of T cells and the production of cytokines.The expression of PD‐L1 is a predictive biomarker for immunotherapy. The original selective criteria of immunotherapy for advanced NSCLC patients were set as patients with PD‐L1 expression in tumor tissue ≥ 50%. In the trial supporting nivolumab for squamous NSCLC, the outcomes were not assumed to be associated with PD‐L1 expression status.5.2.1EGFR or ALK mutations were approved by the U.S. FDA.Nivolumab (Opdivo), a human immunoglobulin G4 (IgG4) mAb targeting human PD‐1, binds PD‐1 with high affinity and blocks the interaction between PD‐1 and PD‐L1/PD‐L2.Pembrolizumab , a highly selective IgG4‐κ isotype mAb against PD‐1, blocks PD‐1/PD‐L1/PD‐L2 pathway via binding to PD‐1.Cemiplimab‐rwlc is the third anti‐PD‐1 antibody approved by the U.S. FDA for the treatment of advanced NSCLC patients with PD‐L1 expression of at least 50%. Based on the results from Study 1624, cemiplimab‐rwlc showed significantly improved PFS (6.2 vs. 5.6 months) and OS (22.1 vs. 14.3 months) compared to those treated with platinum‐based chemotherapy.There are other three anti‐PD‐1 antibodies approved by NMPA, not by the U.S. FDA, including sintilimab, camrelizumab, and tislelizumab, for the first‐line treatment of NSCLC. The indications of these three ICIs were all referred to combination with chemotherapy for advanced NSCLC with any PD‐L1 expression level. In 2020, based on the data from a phase III trial, ORIENT‐11, sintilimab in combination with pemetrexed and platinum‐based chemotherapy was approved by NMPA for the first‐line treatment of advanced nonsquamous NSCLC.Camrelizumab is an anti‐PD‐1 antibody approved in China, which has the most indications in antitumor therapy, including classic Hodgkin lymphoma,Tislelizumab was approved for the first‐line treatment of nonsquamous NSCLC in 2020, which was based on the data from a clinical trial, RATIONALE 304.5.2.2The treatment strategy for targeting PD‐L1 is similar to anti‐PD‐1, but still has the interaction between PD‐1 and PD‐L2, which is assumed to help balance the body inflammatory response.EGFR or ALK mutation.Atezolizumab (MPDL3280A), a human IgG1 mAb, is the first anti‐PD‐L1 antibody approved for NSCLC patients. In 2016, atezolizumab was initially approved as a second‐line treatment for advanced NSCLC patients whose disease has progressed after chemotherapy.Durvalumab (MEDI4736) is a human IgG1 antibody with high affinity of PD‐L1, which was approved by the U.S. FDA as adjuvant treatment of unresectable advanced NSCLC, whose disease has not progressed following concurrent platinum‐based chemotherapy and radiation therapy. This approval was based on the data from a phase III trial, PACIFIC (NCT02125461). Advanced NSCLC completed concurrent chemotherapy and radiation and then received durvalumab or placebo. The PFS in durvalumab group was significantly improved compared with placebo arm.5.3CTLA‐4 is another inhibitory molecule expressed on activated T cells to block the excessive immune response. The responding ligands for CTLA‐4 include CD80 (B7.1) and CD86 (B7.2), which are similar to CD28, an activating signal molecule on T cell surface.5.3.1EGFR or ALK mutation. The OS of patients in immunotherapy combination arm was 17.1 versus 14.9 months in chemotherapy arm. Of note, the combination of two immunotherapy agents demonstrated more treatment‐related adverse events than single agent, with 76.7% of patients reported.Ipilimumab (BMS‐734106) is a fully humanized IgG1 mAb and was first approved in melanoma treatment with favorable outcomes.5.4EGFR or ALK genomic tumor aberrations. In late 2021, tiragolumab in combination with dabrafenib, a BRAF TKI, was approved by the U.S. FDA for the treatment of metastatic NSCLC with BRAF V600E mutation. This approval was based on the data from Study BRF113928 (NCT01336634), in which the ORRs in previously treated and untreated group were 63% and 61%, respectively, compared with 27% of dabrafenib single‐agent group.Tiragolumab is an anti‐TIGIT mAb. The data of combination of tiragolumab with atezolizumab in CITYSCAPE study have been released at the ASCO2020 conference. Addition of tiragolumab to atezolizumab demonstrated an increased RR (37.3% vs. 20.6%) and PFS (5.6 vs. 3.9 months) versus atezolizumab alone.6The rapid development of targeted therapy and immunotherapy has profoundly changed the treatment strategy for NSCLC. Balancing the benefits and risks of various treatments and providing patients with best treatment with less adverse events is important for the treatment decisions. Diversity of combinations have been investigated in clinical trials.6.1There is limited evidence supporting the efficacy of combination of chemotherapy and targeted therapy. A phase II clinical trial found a PFS benefit (15.8 vs. 10.9 months) of gefitinib plus pemetrexed compared with gefitinib alone, but without statistical significance.6.2EGFR or ALK mutations, according to the current evidence. In AXL gene aberration‐positive NSCLC patients, bemcentinib, an AXL TKI, in combination with pembrolizumab showed significantly improved prognosis and has been granted fast track designation by the U.S. FDA in June 2021 (NCT03184558). Therefore, targeted therapy in combination with immunotherapy might be an alteration for drug development of those immature targets. However, for patients get drug resistance and progress on targeted therapy, immunotherapy plus chemotherapy and antiangiogenesis is a promising strategy to improve survival.EGFR mutation‐positive NSCLC patients, with improved PFS (10.2 vs. 7.1 months) in atezolizumab plus bevacizumab and chemotherapy group compared with bevacizumab and chemotherapy combination group in EGFR mutation‐positive NSCLC patients.EGFR mutation‐positive advanced NSCLC.Generally, immunotherapy is less effective than targeted therapy in NSCLC patients with targetable driver gene mutations. Several clinical trials evaluated the efficacy of immunotherapy in previously treated NSCLC patients with driver gene mutations.6.3EGFR or ALK mutations. Based on the data from clinical trials, different chemoimmunotherapies have been approved by the U.S. FDA for the first‐line treatment of advanced NSCLC.The combination of immunotherapy and chemotherapy is a new standard therapeutic regimen for advanced NSCLC patients, especially those without driver gene mutation. The basic theory is that increased expression of tumor antigens and PD‐L1 expression in the immunological environment induced by chemotherapy agents might enhance the therapeutic effects of immunotherapy.KeyNote 189 is the first clinical trial demonstrating the promising therapeutic effects of chemoimmunotherapy compared with chemotherapy alone in nonsquamous NSCLC, in which pembrolizumab in combination with platinum‐based chemotherapy significantly improved the OS and PFS of NSCLC patients without EGFR or ALK mutations.Studies also have been performed in squamous NSCLC and demonstrated promising outcomes. KeyNote 407 is the first phase III study to make a change in the standard treatment for squamous NSCLC. Carboplatin–paclitaxel or abraxane in combination with pembrolizumab demonstrated a better OS (15.9 vs. 11.3 months) compared with chemotherapy alone.6.4The efficacy of combination of anti‐PD‐1/PD‐L1 pathway with anti‐CTLA4 has been under clinical investigation. As mentioned above, nivolumab in combination with ipilimumab has been approved by the U.S. FDA for the first‐line treatment of advanced NSCLC.6.5EGFR or ALK genomic tumor aberrations. In 2020, the U.S. FDA approved the first anti‐VEGFR and EGFR–TKI combination as first‐line treatment for advanced NSCLC. Ramucirumab (Cyramza), a VEGFR2 antagonist, in combination with erlotinib showed improved survival for patients with EGFR exon 19 deletion and L858R mutations. But patients in combination group also demonstrated high incidence (72%) of grade 3–4 treatment‐emergent adverse events.There are two antiangiogenic drugs approved by the U.S. FDA for the treatment of NSCLC. Bevacizumab in combination with chemotherapy (carboplatin and paclitaxel) was initially approved by the U.S. FDA in 2006, for the treatment of advanced nonsquamous NSCLC.77.1HER2 mutations.EGFR insertion and HER2 mutation, which is approved for the treatment of EGFR exon 20 insertion mutations. Mobocertinib is granted a BTD from the U.S. FDA for NSCLC with EGFR or HER2 exon 20 insertion mutations, based on the data from a phase II trial.HER2‐mutated NSCLC in a phase II trial, ZENTITH20‐2.HER2 and HER3 belong to an RTK family that includes EGFR (ERBB1), HER2 (ERBB2/NEU), HER3 (ERBB3), and HER4 (ERBB4). In contrast to EGFR, no ligand has been identified for HER2 and only one ligand, heregulin (HRG), for HER3 has been identified. Still, they are involved in receptor interaction and promote the dimerization of all ERBB family components.EGFR mutation express HER3.EGFR mutations in a phase I clinical trial, with a disease control rate of 70% and median duration of 6.9 months.The aberrant activation of HER3 is observed in NSCLC and more than 80% of NSCLC patients with 7.2phosphatidylinositol‐4,5‐bisphosphate 3‐kinase, catalytic subunit alpha (PIK3CA) gene encodes the catalytic subunit alpha of PIK3. However, the mutations of PIK3CA are not common in lung cancer (mainly observed in LUSC), by contrast amplification of which is more prevalent.PIK3CA mutations are associated with worse OS and PFS in patients with NSCLC.The phosphoinositide 3‐kinase (PI3K) pathway regulates multiple cellular biologic process. The aberrant activation of PI3K/AKT/mTOR signaling is common in different cancer types.7.3Epigenetic alteration is one of the cancer hallmarks, which is mainly referred to DNA methylation, histone deacetylation, and noncoding RNAs (ncRNAs). Epigenetic drugs have made breakthroughs in hematologic malignancies and are approved by the U.S. FDA, including HDAC inhibitors for cutaneous T cell lymphoma (vorinostat and romidepsin), DNMT inhibitors for myelodysplastic syndrome (5‐azacytidine and decitabine), and JAK1/2 inhibitor for myelofibrosis (ruxolitinib). The approval of tazemetostat, an EZH2 inhibitor, for epithelioid sarcomas is a milestone in epigenetic anticancer therapy for solid tumor.DNA hypermethylation happens during early stage of carcinogenesis and has been widely studied.APC, RASSF1A, CDH13, and CDKN2A.The treatment strategy through regulation of epigenetic events is usually based on combination therapy.8Surgery provides the best chance to cure the disease, but most NSCLC patients present with advanced‐stage disease. Cytotoxic chemotherapy had held a leading role in the treatment of advanced NSCLC until the first time TKIs became the first‐line treatment in certain subgroups of NSCLC patients. During the last decade, targeted therapy and immunotherapy have changed the treatment strategies for NSCLC, especially those in late phases of this disease. Based on the features of genetic alterations and PD‐L1 expression, clinicians make the final treatment decision more personalized. However, major challenges still remain, including identifying new targetable genetic alterations, developing new drugs and effective drug combinations, overcoming drug resistance, and discovering better biomakers to predict therapeutic response and prognosis of patients. Combination therapy could be an effective strategy in overcoming TKI resistance by targeting the native genomic mutation and the secondary alterations. However, the use of combination therapies increases the drug‐associated toxicity, which requires strict validation. To determine further treatment, molecular testing is not only essential for initiation of treatment, but also necessary at the time that disease progresses.Currently, a variety of treatment strategies are available for NSCLC. Choosing appropriate and personalized combination therapy for patients is challenging for clinicians. Of note, patients not only benefit from the release of new drugs, but also benefit from new use or combos of the existing drugs.The authors declare no competing interests.Ethics approval was not required for this review.Yuan Cheng: conceptualization, methodology, and writing. Tao Zhang: Figure preparation. Qing Xu: conceptualization and reviewing.


Wilson disease is a rare, disabling, neurological genetic disease. Biomarkers of brain damage are less well developed.The aim of this study was to evaluate the utility of plasma glial fibrillary acidic protein as a biomarker for neurological involvement in patients with Wilson disease.This prospective cross‐observational study compared plasma glial fibrillary acidic protein concentration among different subtypes of patients with Wilson disease and healthy control subjects. Plasma glial fibrillary acidic protein levels were measured in 94 patients and 25 healthy control subjects. Patients were divided into two subtypes: patients with neurological manifestations (n = 74) or hepatic manifestations (n = 20).Median levels of plasma glial fibrillary acidic protein were significantly elevated in patients with neurological manifestations (143.87 pg/mL) compared with those with hepatic manifestations (107.50 pg/mL) and healthy control subjects (86.85 pg/mL). Receiver operating characteristic curve revealed that a plasma glial fibrillary acidic protein cutoff value of 128.8 pg/mL provides sufficient sensitivity (80.0%) and specificity (63.5%) to differentiate patients with neurological manifestations from those with hepatic manifestations.Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyPlasma glial fibrillary acidic protein may serve as a biomarker for distinguishing different subtypes of Wilson disease. © 2021 The Authors.  ATP7B gene, which is mapped to chromosome 13 (13q14.3), encodes a transmembrane copper‐transporting P‐type ATPase.ATP7B lead to copper overload in organs, predominantly the liver and the brain, resulting in the occurrence of hepatic and neurological manifestations.Wilson disease (WD) is a rare autosomal recessive inherited disorder of copper metabolism, with a genetic prevalence of approximately 13.9 per 100,000 individuals.Although rare, WD can be managed successfully if recognized early, diagnosed accurately, and treated correctly.Long‐term exposure to high copper concentrations ultimately results in damaged astrocytes and dysfunction of the blood–brain barrier.A biomarker that clearly differentiates neurological from nonneurological WD would likely have substantial utility. For example, it could be used to predict prognosis or as a measure of relevant disease activity or target engagement in clinical trials. Hence we examined the levels of plasma GFAP (pGFAP) among patients with WD to investigate whether GFAP values were correlated with the presence of neurological involvement and disease severity.These genetically confirmed patients were recruited from a WD cohort study  at the Neurogenetic Diseases Center in the First Affiliated Hospital of Fujian Medical University and were consecutively enrolled between July 1, 2019, and June 1, 2020. A diagnosis of WD was established based on typical symptoms and clinical, biochemical, and genetic findings. A positive diagnosis for WD was determined by a Leipzig score of ≥4.Patients were divided into two subgroups: WD‐NM, defined as the presence of typical neurological symptoms regardless of whether they were accompanied by hepatic abnormalities; and WD with hepatic manifestations (WD‐HM), defined as the absence of neurological symptoms and the presence of hepatic abnormalities, typically characterized by abnormal liver function test, diffuse lesions of the liver parenchyma observed in ultrasound tests, or symptoms of cirrhosis.We further recruited 25 healthy control subjects , all of whom appeared to have normal brain MRI results. The study protocol and informed consent procedures were approved by the institutional review boards and ethics committee at First Affiliated Hospital of Fujian Medical University. Written informed consent was provided by all participants.ATP7B gene were performed as previously reported.Direct sequencing of all exons and flanking regions in the All patients' medical records and brain MRI data were collected. An experienced neurologist collected demographic information and clinical characteristics by questionnaire, including age at onset, initial symptoms, disease duration, assistant examination, current medications, and Unified Wilson's Disease Rating Scale (UWDRS) at the same time as plasma collection. Neurological symptom severity was evaluated by UWDRS, including functional impairment , neurological examination , and psychiatric assessment .g for 10 minutes at 20°C, with subsequent storage of plasma aliquots in cryogenic vials at −80°C until use. pGFAP levels were measured using a commercial Simoa GFAP Discovery Kit  on a Simoa HD‐1 Analyzer instrument, according to the manufacturer's instructions. Plasma samples were diluted at a ratio of 1:4, and the detection range of the kit was 0–1000 pg/mL. Samples were anonymized by assignment of random numbers to replace identifying information, and measurement was performed by technicians with no knowledge of the experiment.Blood sampling was conducted by collecting 10 mL venous blood in EDTA‐containing tubes, followed by centrifugation at 3500t test, and variables that violated the assumptions of normality were analyzed by the Mann–Whitney U test. Categorical variables were compared by chi‐square test and Fisher exact test. Abnormal GFAP values were defined as pGFAP concentrations greater than 132.48 pg/mL . Multiple regression analysis was used to eliminate potentially confounding factors  that may influence pGFAP levels. Receiver operating characteristic (ROC) curve analyses were performed to assess diagnostic sensitivity and specificity of pGFAP detection. We used a linear regression model to explore associations between pGFAP concentration and disease severity. Statistical analysis was performed using SPSS version 26.0 software  and GraphPad Prism version 8.3 software . P < 0.05 was considered statistically significant.Categorical variables were described by counts and percentages, while continuous and ordinal variables were described by median and interquartile ranges. Continuous variables that followed normal distributions were compared using P < 0.05]. The groups showed no significant difference in sex ratio.Demographic and clinical data for all patients are listed in Table P = 0.0045, Mann–Whitney U test] and HCs   after adjusting for sex, age, age at onset, and different treatments. To assess the ability of pGFAP to distinguish between patients with WD‐NM and HCs, we performed ROC curve analysis, which showed that a pGFAP cutoff value of 93.25 pg/mL resulted in 89.2% sensitivity and 72.0% specificity, with an area under the curve of 0.864  pg/mL] were greater than in patients with WD‐HM  Fig. . After a864 Fig. . The ROC864 Fig. .P = 0.954), UWDRS‐N subscore , or UWDRS‐P subscore    Fig. . Nor was85) Fig. .In addition, we observed the clinical characteristics of two patients with WD‐HM who had abnormal levels of pGFAP. Case 1, an 8‐year‐old boy, was found to exhibit abnormal liver function during a medical checkup at the age of 1 year. From that time until present, he was kept on a low‐copper diet with daily supplements of zinc salts and regular follow‐up examinations. His MRI was normal, ultrasound showed echogenic liver parenchyma, and 24‐hour urinary copper level was 82.03 μg during the study. Case 2, a 29‐year‐old woman, initially sought medical advice for edema of both legs at age 13 years and took zinc salts daily for an extended period. Her MRI was normal, ultrasound showed echogenicity of liver parenchyma with splenomegaly, and her 24‐hour urinary copper level was 69.43 μg during the study.Previous studies have reported that pGFAP concentration is related to brain injury.We also explored the correlation between pGFAP levels and disease severity, that is, clinical symptoms and MRI lesions. Our results showed that pGFAP levels were not correlated with disease severity. Two reasons at least partially explain this result. Disease severity is a function of long‐term damage accumulation in WD, which is a cumulative value. Thus, pGFAP concentration at a specific time point may not reflect general brain damage. Moreover, a nonlinear natural history of CNS injury progression in WD may contribute to fluctuations in pGFAP concentration that prevent a continuous correlation between disease severity and pGFAP.Given the rarity of the longitudinal data in this study, we could not definitively determine whether pGFAP is consistent and reliable as a biomarker for disease severity, disease activity, and treatment response on neurological injury in WD. The cohort size is small, and the sensitivity and specificity of the ROC analysis of GFAP levels are not very robust, so the results of this study should be interpreted with caution. Nevertheless, the results of this study may urge further evaluation by large‐scale clinical trials to explore biomarkers for distinguishing WD‐NM from WD‐HM.Y.F., N.W., and W.‐J.C. conceived the study concept and design. J.L., Y.Z., Yi Lin, X.‐H.L., and W.‐H.L. acquired the data. Laboratory determination was done by W.Z. Y.F. and Ying Liu. analyzed the data. J.L. and Q.W. performed the statistical analysis. Y.F. and J.L. drafted the manuscript.All authors declare that there are no additional disclosures to report.The study was approved by the First Affiliated Hospital of Fujian Medical University institutional review boards and ethics committee.


Circumstantial evidence indicates that aerosol transmission is a likely contributor to the current pandemic,Despite the demonstrated challenges in capturing suspended live virus, respiratory transmission is now thought of as the primary mode of SARS‐CoV‐2 infection. By contrast, SARS‐CoV‐2 has been readily cultured from nasopharyngeal (NP) swabs, saliva, blood, stool, and semen.Building on previous work carried out by our team,From October 2020 to January 2021, we visited eight patients in their homes in and around Durham, North Carolina soon after they were confirmed by molecular assay to be infected with SARS‐CoV‐2. After informed consent was obtained, we asked patients to complete a brief questionnaire, and to permit the collection of a NP swab, passive saliva sample, fomite swabs, and bioaerosol samples. Patients were also asked to self‐collect a rectal swab sample. All study procedures were approved by the Duke University Institutional Review Board (Pro00105055).air/min.air/min. These samplers are designed to capture viral matter in liquid media to enhance viability. It is important to note that we have previously used both types of samplers to capture live influenza A virus.Bioaerosol sampling was carried out using National Institute for Occupational Safety and Health (NIOSH) BC 251 aerosol samplers, placed ~1.5 meters off the ground at distances of ~1 meter, 1.4 meters, 2.2 meters, and 3.2 meters from the participant's head. The participants were asked to remain stationary in their room and the samplers were run for approximately two hours at a calibrated flow‐rate of 3.5 LBiological samples and fomite swabs were collected and processed as previously described,Viral RNA was extracted from processed samples using QIAamp Viral RNA Mini Kits , with the resultant product used in an adapted Center for Disease Control and Prevention (CDC) 2019‐nCoV real‐time RT‐PCR assay.All participants presented with mild to moderate illness. The majority of the participants were females . The predominantly represented race was white , with two Black participants (25.0%), and one Asian (12.5%). One participant identified as Hispanic, and a second as Indian. The mean age was 41.4 years, with a range of 29 to 53 years. Among all participants, five lived in a private house, one in an apartment complex, and two in a residential shelter.Participants were typically enrolled within three days of symptom onset (one patient was enrolled on day 8), and reported experiencing between zero and seven common COVID‐19 symptoms at time of enrollment, with cough, fatigue, and body ache the most frequently listed Table . ChronicSampling sites in participants’ homes varied in size, with some permitting as few as 6 total bioaerosol samplers and others as many as 10 samplers. Among the eight participants, a total of 139 samples were collected. Among those, 45 (32.4%) were SARS‐CoV‐2‐positive by RT‐PCR, including 20 biological samples , 12 fomites , and 13 bioaerosol samplers  .None declared.Emily R. Robie: Data curation (lead); Investigation ; Project administration (supporting); Writing‐original draft (lead); Writing‐review & editing . Anfal Abdelgadir: Investigation ; Writing‐review & editing . Raquel A. Binder: Investigation ; Supervision (supporting); Writing‐review & editing . Gregory C. Gray: Conceptualization (lead); Funding acquisition (lead); Investigation ; Methodology (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing‐review & editing .https://publons.com/publon/10.1111/irv.12860.The peer review history for this article is available at Table S1Click here for additional data file.


The anthracycline drug doxorubicin is an effective anticancer drugs with both DNA‐ and chromatin‐damaging activity. While the chromatin‐damaging activity constitutes major anticancer efficacy of doxorubicin, combination with the DNA‐damaging activity plagues the drug with long‐term toxicities such as cardiotoxicity, therapy‐related malignancies and gonadotoxicity. Therefore, developing DNA damage‐free anthracyclines is a promising direction for novel treatment options with limited side effects, which has been shown to be possible. The anthracycline drug doxorubicin is among the most used—and useful—chemotherapeutics. While doxorubicin is highly effective in the treatment of various hematopoietic malignancies and solid tumours, its application is limited by severe adverse effects, including irreversible cardiotoxicity, therapy‐related malignancies and gonadotoxicity. This continues to motivate investigation into the mechanisms of anthracycline activities and toxicities, with the aim to overcome the latter without sacrificing the former. It has long been appreciated that doxorubicin causes DNA double‐strand breaks due to poisoning topoisomerase II. More recently, it became clear that doxorubicin also leads to chromatin damage achieved through eviction of histones from select sites in the genome. Evaluation of these activities in various anthracycline analogues has revealed that chromatin damage makes a major contribution to the efficacy of anthracycline drugs. Furthermore, the DNA‐damaging effect conspires with chromatin damage to cause a number of adverse effects. Structure–activity relationships within the anthracycline family offer opportunities for chemical separation of these activities towards development of effective analogues with limited adverse effects. In this review, we elaborate on our current understanding of the different activities of doxorubicin and their contributions to drug efficacy and side effects. We then offer our perspective on how the activities of this old anticancer drug can be amended in new ways to benefit cancer patients, by providing effective treatment with improved quality of life. AMLacute myeloid leukaemiaAPLacute promyelocytic leukaemiaDAMPsdamage‐associated molecular patternsDSBsdouble‐strand breaks2O2Hhydrogen peroxideICDimmunogenic cell deathdiMe‐doxorubicinN,N‐dimethyl‐doxorubicinO2·‐superoxide anionROSreactive oxygen speciest‐AMLtherapy‐related acute myeloid leukaemiat‐APLtherapy‐related acute promyelocytic leukaemiat‐MNstherapy‐related malignant neoplasmsTopo IItopoisomerase IITopo IIαtopoisomerase II alphaTopo IIβtopoisomerase II betaStreptomyces peucetius [S. peucetius [Doxorubicin, also known as adriamycin, is a member of the anthracycline anticancer drug family Fig. . The fireucetius . Soon, ieucetius , 4. In 1eucetius . Doxorubeucetius , 7. Howeeucetius . Cardioteucetius , 10. As eucetius , 11. WitThe classical mechanism of action by which anthracyclines function is inhibition or poisoning of Topoisomerase II (Topo II) . This enAnthracyclines intercalate into the DNA helix with their anthraquinone moiety. While rings B and C of the tetracycline moiety overlap with adjacent DNA base pairs, and ring D passes through the intercalation site, the sugar moiety is pointed into the minor groove, which may compete for space with histones , 27. In O2·‐) and hydrogen peroxide (H2O2), or oxidize the bond between the sugar and the aglycon resulting in reductive deglycosylation. Eventually O2·‐, and H2O2 are converted into more reactive hydroxyl radicals (·OH) via the iron‐catalysed Haber‐Weiss reaction [The quinone moiety in ring C of anthracyclines can be transformed into a semiquinone by a number of oxidoreductases, including cytochrome P450 reductases, xanthine oxidase and NADH dehydrogenase (complex I) of the mitochondrial electron transport chain , 31. Subreaction , 33. In reaction , 35, 36.reaction . NotwithIn vitro experiments with reconstituted single nucleosomes showed that doxorubicin causes nucleosome dissociation in an ATP‐, transcription‐ and histone chaperone‐independent manner, which may explain why the structure of Topo II‐DNA‐doxorubicin complex is not available [To organize two metres of DNA in the nucleus of a single cell, DNA is compacted at several levels. One level of organization is the formation of nucleosomes, where a segment of 146 base pairs of DNA is wrapped around eight histone proteins . As mentvailable . Moreovevailable , 40. Thevailable ) or otheThe dynamic structure of chromatin is essential for many nuclear processes, including transcription and replication. Therefore, the assembly, spatial organizing and compactization of chromatin are tightly regulated by various histone chaperones, ATP‐dependent chromatin remodelling complexes and histone‐modifying enzymes , 42. BeiN,N‐dimethyl‐doxorubicin (diMe‐doxorubicin), which induce histone eviction without generating DNA damage [How histone eviction exactly causes cell death remains unclear, but it is likely to play a major contribution to the anticancer activity of the anthracycline drugs. This is illustrated by the anthracycline drugs aclarubicin and A damage . AclarubA damage , 39, 44.A damage . It exhiA damage , 39, 44.A damage , but no + T‐cell response in a colon carcinoma mouse model [Besides the direct effect on eliminating tumour cells, anthracyclines can also promote antitumour immunity. During cell death, cell contents can be released into the tumour microenvironment, including tumour antigens and danger signals  . These Dse model . Moreovese model . Recentlse model . So it wse model . AlthougAlthough doxorubicin has been a cornerstone in cancer treatment for nearly five decades, its use is plagued with severe and treatment‐limiting side effects. Next to common generally acute and reversible chemo‐related adverse effects, such as nausea, vomiting, diarrhoea and bone marrow suppression, anthracycline treatment is associated with long‐term side effects, namely cardiotoxicity, therapy‐related malignancies and gonadotoxicity. These long‐term adverse effects severely impact the quality of life of cancer survivors, which limit the further application of anthracyclines. Therefore, extensive research has been performed to understand and reduce the anthracycline‐induced long‐term side effects.−2 [The most treatment‐limiting and therefore probably the best studied side effect of anthracyclines is cardiotoxicity. Anthracycline‐induced cardiotoxicity presents as cardiomyopathy, ventricular dysfunction, pericarditis‐myocarditis syndrome or arrhythmias and is dose‐dependent and irreversible , 55, 56.−2 , 10. Bes−2 , 58. CurMultiple mechanisms have been proposed, including mitochondrial dysfunction and/or lipid peroxidation as a result of ROS formation, targeting Topo II beta (Topo IIβ) in cardiomyocytes, and effects on calcium homeostasis , 61, 62.et al. [The most intensely studied mechanism of anthracycline‐induced cardiotoxicity is ROS production through interference with redox cycling and mitochondrial function . To meetet al.  showed tet al. . Althouget al. , 67.Similar to ROS scavengers, most iron‐chelating agents can reduce ROS formation and alleviate doxorubicin‐induced cardiotoxicity in preclinical models. However, such benefits were not observed in patients , 69. Thein vitro studies, the discrepancy between the effectivity of ROS scavengers and iron chelators in preclinical studies and patients challenges the contribution of ROS production in anthracycline‐induced heart damage. Using appropriate preclinical cardiotoxicity models and treatment with anthracyclines at clinical relevant concentrations and schedules may help clarifying this issue.Although it is convincingly shown that anthracyclines can induce ROS formation in et al. [+/Δ and Topo IIβΔ/Δ) were not susceptible to the cardiac impairment caused by doxorubicin as observed in Topo IIβ+/+ mice. Further, Lyu et al. [in vitro by rapid proteasomal degradation of Topo IIβ. These studies indicate that the DSBs mediated by Topo IIβ poisoning is a major cause of doxorubicin‐induced cardiotoxicity. Nevertheless, DSB cannot be the only reason, since the structurally nonrelated Topo II poison etoposide does not cause cardiotoxicity. From a clinical point of view, it suggests that Topo IIα‐specific anthracycline would prevent cardiotoxicity in patients and that Topo IIβ expression could be used as a prognostic marker for cardiotoxicity. Unfortunately, no genuine Topo IIα‐ or Topo IIβ‐specific drugs are available in clinic at present.In human, Topo II enzymes are expressed in two isoforms, Topo IIα and Topo IIβ . Althouget al.  reportedu et al.  reportedDue to the unsatisfactory effects of ROS scavengers and iron chelators in the clinic, tumour‐specific drug delivery systems were introduced in 1990s to reduce doxorubicin‐induced toxicities. These delivery strategies included nanoparticle encapsulated liposomal doxorubicin (LD) and pegylated LD (PLD). LD and PLD both show prolonged serum half‐life and a smaller volume of distribution compared with conventional doxorubicin . LD and P = 0.008) and subclinical cardiotoxicity  without compromising antitumour efficacy [−2) compared with doxorubicin (450–550 mg·m−2). Although epirubicin can be used at higher cumulative dose, its application is still limited by cardiotoxicity.With the aim to identify more effective anthracyclines with fewer side effects, thousands of doxorubicin analogues, either isolated from natural sources, produced by mutant enzymes or prepared by organic (semi)synthesis, have been evaluated in the past decades. However, only few variant drugs showed reduced cardiotoxicity without loss of anticancer activity. One such analogue which entered the clinic is epirubicin. In a meta‐analysis, epirubicin treatment showed significantly less cardiotoxicity compared with doxorubicin , which determines whether it is theoretically feasible to eliminate the cardiotoxicity of anthracycline without compromising its therapeutic efficacy. Recent work of our group provides some insight. We observed that aclarubicin, as well as the doxorubicin analogue diMe‐doxorubicin, showed strongly reduced cardiotoxicity in various mouse models and human‐induced pluripotent stem cells‐derived cardiomyocyte microtissues, without compromising anticancer activity . N,N‐dimity Fig. . On the in vitro and tumorigenic in vivo [Attributing to the increased survival of cancer patients which modern anticancer therapy has made possible, the long‐term side effects, such as tumorigenicity, have become an issue. Currently, 17–19% of all new primary malignancies occur in cancer survivors , 83. Amo in vivo , 89, 90, in vivo , 92, 93, in vivo , 95, 96  in vivo . Thyroid in vivo  and acut in vivo , 100 havMLL1 gene) or 21q22 (involving AML1/RUNX1/CBFA2 gene), however occurring at unique breakpoints than de novo AML with the same cytogenetics [de novo AML [de novo t APL, the clinical outcomes of t‐APL and de novo APL are similar after all‐trans retinoic acid‐ and anthracycline‐based treatments, for which the 5‐year survival rate is about 80% [+/−Trp53 female mice, indicating the direct contribution of doxorubicin treatment to tumour development [The anthracycline therapy‐related AMLs (t‐AMLs) frequently exhibit balanced chromosomal translocations at 11q23 , carcinogenic exposures in common , host effects  and combination therapy with other mutagenic chemotherapeutics  , 96, 97.herapies . This tuherapies .+/−Trp53 mouse experiment and in vitro data showed that aclarubicin and diMe‐doxorubicin without DNA‐damaging activity are not tumorigenic [H2AX is an important histone variant for DNA damage repair, which is phosphorylated at DNA damage sites and responsible for repair machinery recruitment. Eviction of H2AX by doxorubicin greatly attenuates DNA damage repair and consequently results in enhanced cell death and more transformation compared with etoposide , 43. In origenic , 118. CoMLL1 translocation is also associated with epigenetic changes, since MLL1 is an H3K4 methyltransferase [MLL1–partner‐3′ rearrangement. Epigenetic profiling after MLL1 deletion or with MLL1 fusion proteins revealed reduced H3K4 methylation at promotor region of target genes [As mentioned above, anthracyclines evict histones with different epigenomic selectivity. It is interesting to notice that t(11q23) AML with nsferase . The C‐tet genes , 121. Coet genes , 123. Aset genes , 128.Due to limited understanding of the mechanisms of action, t‐MN was previously considered as the original sin of anthracycline treatment because of resulted DNA damage. Hence, hope was laid on early detection of t‐MNs by intense follow‐up screening in susceptible cancer survivors or restraint of high cumulative dose of anthracyclines. However, the discovery of histone eviction activity of anthracyclines not only offers a new anticancer mechanism, but also provides a strategy to prevent t‐MNs, which is experimentally illustrated by aclarubicin , 117. ThOwing to its mechanisms, doxorubicin also targets healthy tissues with high proliferating rates, such as myeloid and lymphoid tissues, gastrointestinal mucosa and gonads. Since the survival rates of cancer patients improved spectacularly in the last two decades, the number of cancer survivors suffering from doxorubicin‐induced gonadotoxicity also strongly increased . Gonadotin vivo. Nevertheless, development of active anthracycline variants with limited gonadotoxicity would be a preferable strategy, if possible. The depletion of follicular reserve in females and depletion of spermatogenesis in males caused by doxorubicin treatment can be attributed to the DSBs generated by the drug and subsequent cell death of germ cells [Several classes of compounds have been proposed to protect gonads from doxorubicin insult in mouse models, including hormone agonists , anti‐oxrm cells , 144. Berm cells , 147. Thrm cells , 149. Thin vivo [Oxidative stress has also been proposed as a mechanism of doxorubicin‐induced gonadotoxicity . Howeverin vivo , 152. CoSince the discovery of daunorubicin and doxorubicin in the 1960s, a search for less toxic yet effective alternatives to doxorubicin was initiated in the 1980s. Out of thousands of anthracycline variants tested, only a few entered the clinic, most notably epirubicin, idarubicin and aclarubicin. One reason for this limited number of successful compounds might be the lack of consensus on the mechanism of action of anthracyclines for their anticancer activity and toxicities. Furthermore, whether the severe toxicities of these drugs are intimately connected with their anticancer activity has been a lingering topic in the field. For a long time, DSB induction was considered as the main anticancer activity of anthracyclines. While only recently, a second activity –chromatin damage as a result of histone eviction– was proposed , 39. ChrWhile the potential cardiotoxicity‐low/free anthracyclines need to be tested in clinic, some improvements of current anthracycline‐containing chemotherapy regimen should be considered. For instance, it would be debatable to combine anthracyclines with etoposide in the same treatment regimen concerning the contribution of DNA‐damaging activity to multiple toxicities, although this is frequently used in AML treatment. Likewise, specific anthracycline variant should be carefully selected for children cancer patients or patients with predisposal genetic disorder to avoid toxicities. The new mechanism, histone eviction with certain (epi)genomic selectivity, indicates that anthracyclines are in fact also epigenetic drugs. Preliminary data showed that diffuse large B‐cell lymphoma cells with elevated levels of H3K27me3 were more susceptible to aclarubicin than daunorubicin , indicatThe recent understanding on anthracycline anticancer activity and toxicities suggests that anthracycline development should focus on depleting DNA‐damaging activity from chromatin‐damaging activity. Such drugs should allow effective anthracycline‐based therapies devoid of the major treatment‐limiting adverse effects: cardiotoxicity, therapy‐related malignancies and gonadotoxicity. This would especially benefit cancer patients with a poor heart function, which are currently excluded from anthracycline‐based chemotherapy. In addition, drug variants lacking these side effects could be used in more intense and/or longer therapy and could be used for relapsed patients with a history of anthracyclines‐based therapies.In conclusion, despite the long history of anthracyclines, the novel discovery of chromatin damage as the major antitumour activity and its collective contribution with DNA‐damaging activity to toxicities, allows the development of potentially new treatment strategies to improve cancer therapy and the quality of life of cancer survivors.JN is a shareholder in NIHM that aims to produce aclarubicin for clinical use.SvdZ and XQ conceived the manuscript and constructed the text under supervision of JN.https://publons.com/publon/10.1111/febs.15583.The peer review history for this article is available at


Identification of a urinary metabolite biomarker with diagnostic or prognostic significance for early immunoglobulin A nephropathy (IgAN) is needed. We performed nuclear magnetic resonance‐based metabolomic profiling and identified 26 metabolites in urine samples. We collected urine samples from 201, 77, 47, 36 and 136 patients with IgAN, patients with membranous nephropathy, patients with minimal change disease, patients with lupus nephritis and healthy controls, respectively. We determined whether a metabolite level is associated with the prognosis of IgAN through Cox regression and continuous net reclassification improvement (cNRI). Finally, in vitro experiments with human kidney tubular epithelial cells (hTECs) were performed for experimental validation. As the results, the urinary glycine level was higher in the IgAN group than the control groups. A higher urinary glycine level was associated with lower risk of eGFR 30% decline in IgAN patients. The addition of glycine to a predictive model including clinicopathologic information significantly improved the predictive power for the prognosis of IgAN [cNRI 0.72 (0.28‐0.82)]. In hTECs, the addition of glycine ameliorated inflammatory signals induced by tumour necrosis factor‐α. Our study demonstrates that urinary glycine may have diagnostic and prognostic value for IgAN and indicates that urinary glycine is a protective biomarker for IgAN. We first hypothesized that a differentially expressed urinary metabolite biomarker in individuals with early IgAN compared to controls could be identified. Further, we asked whether this biomarker has prognostic value and performed experimental validation to determine the pathophysiologic role of the identified metabolite.22.1This study was conducted according to the Declaration of Helsinki. The institutional review board of Seoul National University Hospital, Seoul, Korea (H‐1601‐076‐732) approved the study. All clinical characteristics and biospecimens were prospectively collected with the approval of the study patients with informed consents.2.22 as we intended to study patients without substantial impairment in their kidney function. In addition, a few prominent outliers regarding urine metabolomic profiles, when identified by visualized principal component analysis plots based on the first and second principal components, were excluded to prevent technical bias. As a clinical outcome, an 30% reduction in eGFR from baseline was used, as most patients had relatively preserved kidney function, and the follow‐up duration was relatively short (median 1.6 years).The kidney biopsies and urine sample collection at the timing of diagnosis were performed between October 2009 and March 2016. Patients with pathologically confirmed IgAN from kidney biopsies were included as the study group. We included patients diagnosed with membranous nephropathy (MN), minimal change disease (MCD) or lupus nephritis (LN) as the disease‐control group. We also constructed a healthy control group, with no evidence of kidney dysfunction, including abnormal urinalysis or reduced estimated glomerular filtration rate (eGFR), and assessed donated urine and blood samples from health screening examinees. The baseline clinical characteristics and biospecimens, including morning random urine samples, were collected at the time of kidney biopsy for initial diagnosis and for health screening examinees at their time of visit. eGFR was calculated with the chronic kidney disease epidemiology collaboration equation.2.3The nuclear magnetic resonance (NMR)‐based urine metabolomic profiling method has been previously published, and details are explained in the Supplemental Methods, Figures 2.4During statistical analysis, a biostatistician was blinded to all names of the metabolites, and the names were revealed at the time of planning further experimental validation to limit confirmation bias.The principal component analysis and partial least squares discriminants analysis were performed by ‘mixOmics’ package in R.U test. The correlations between the clinical characteristics and the urinary metabolite levels were investigated with Spearman's test.We compared the urinary metabolite levels between the IgAN group and another disease or healthy control group with the Mann‐Whitney The predictability for IgAN was assessed by area under curve analysed by receiver operating characteristics (ROC) curve, and we compared the area under curve by DeLong's method with ‘pROC’ package in R.The association between the metabolite values and the risk of poor kidney prognosis, a 30% reduction in eGFR, was determined with panelized spline regression. For those metabolites that had a potential linear association with adverse kidney outcome risks, we performed Cox regression analysis to confirm the prognostic significance. Multivariable models were adjusted for age, sex, baseline eGFR, random urine protein/creatinine ratio, mean arterial pressure and pathologic parameters including the Oxford classification scores, history of angiotensin‐converting enzyme inhibitor or angiotensin II receptor blocker use, and history of immunosuppressive agent use at the time of kidney biopsy to include the variables for the current risk prediction tool for IgAN. We also included sex and crescent scores, which were not included in the suggested prediction tool but might have clinical importance, in the full model.We tested whether the addition of metabolite levels to a prediction model including clinical information improved the predictability analysed by continuous net reclassification improvement (cNRI) with thousands of bootstrapping cycles.P value (0.05/number of comparison) was implemented when identifying differences in urine metabolite levels among the study groups or identifying correlation between the metabolite levels and clinical characteristics. In the prognostic analysis or when comparing model predictive power, conventional two‐sided P value < .05 indicated statistical significance. All statistical analyses were performed with R .In the cNRI analysis, when 95% confidence intervals crossed zero, the difference between the prognostic predictability of the models was interpreted to be non‐significant. A Bonferroni‐adjusted 2.5For immunohistochemistry, we included pathologic slides from kidney biopsies of 12 IgAN cases, 6 controls and 3 MCD and MN cases. The primary antibodies used for staining were for the glycine cleavage system, including T protein, P protein, L protein, and H protein and SHMT 1 and 2 (Abcam). For staining, we cut unstained, paraffin‐embedded tissue sections that were stored at the time of kidney biopsy into 4 μm sections. The sections were deparaffinized in xylene and rehydrated with a descending concentration series of ethanol. Sliced specimens were heated three times in a microwave oven for 5 minutes with 10% citrate buffer solution (pH 6.0) to retrieve the antigen. We blocked the endogenous streptavidin activity with 3% hydrogen peroxide in methanol for 10 minutes at room temperature. Blocking reagent  was used to block non‐specific binding. Images were captured using a light microscope system . Quantification of the immunohistochemical staining was performed with ImageJ . We measured the proportion of the stained area with the same cut‐off thresholds for at least 5 high‐power fields in each sample, and the average value was used to represent the degree of staining of the sample.2.6g for 5 minutes). The recovered hTECs were collected from the pallet and incubated in DMEM/F12  for 4 hours. Floating tubules in the media were retrieved and cultured on collagen‐coated petri dishes  until the establishment of colonies of hTECs in REGM medium  supplemented with 10% foetal bovine serum, 12 mg/mL bovine brain extract, 1 mg/mL hydrocortisone, 10 ng/mL epidermal growth factor, 50 mg/mL gentamycin and 50 ng/mL amphotericin B. hTECs at 2‐3 passages were used in the current study.The unaffected kidney cortices of renal cell carcinoma patients were mechanically dissected. In the dissected cortex, the specimens were minced and digested with Hank's 201 balanced salt solution (HBSS) containing 3 mg/mL collagenase  and incubated at 37°C for 1 hour. The digested cells were washed through a series of sieves  with phosphate‐buffered saline, followed by centrifugation (500 We first tested the expression of target glycine cleavage system molecules identified by the above immunohistochemistry after different dosages of tumour necrosis factor‐α (TNF‐α). The use of TNF‐α to trigger the inflammatory response in hTECs was based on a previous study regarding the tubule pathophysiology of IgAN. After 1 hour of treatment, hTECs were incrementally treated with 2.5, 5.0, 10.0 and 20.0 ng/mL TNF‐α. The dosage resulting in the expression of the target glycine cleavage system molecule (10 ng/mL for 1 hour) was implemented to stimulate hTECs in the following experiments.Second, we treated hTECs with 10 ng/mL TNF‐α for 1 hour, with cotreatment of glycine  in cell culture media with incremental dosages of 2.5, 5.0, 10.0 and 20.0 mmol/L. We investigated the relative expression of inflammation‐related molecules, including intercellular adhesion molecule 1 (ICAM1), p38, phosphor‐p38, p65 and phosphor‐p65, to β‐actin. We also measured glutathione concentrations using a glutathione assay kit , along with the inflammation‐related molecules.Third, we inhibited the target glycine cleavage system molecule (protein H) by siRNA and tested the expression of inflammatory molecules under conditions of TNF‐α induction in hTECs. We transfected the hTECs with siRNA for protein H  according to the manufacturer's protocol. As controls, hTECs treated with scrambled siRNA and hTECs in conditions without TNF‐α induction were tested. Levels of glycine in cell lysates were measured in each condition by the glycine assay kit (Abcam) with mean fluorescence intensity units.For immunoblotting of the inflammatory molecules and glycine cleavage system, 80 μg of proteins was separated by electrophoresis on 10% sodium dodecyl sulphate‐polyacrylamide gels and transferred onto Immobilon‐FL 0.4 μmol/L polyvinylidene difluoride membranes . Proteins were probed with primary antibodies targeting ICAM1 (Thermo Fisher Scientific), p38 (Abcam), phosphor‐p38, p65, phosphor‐p65 , protein H and protein T (Abcam) followed by anti‐rabbit IgG  secondary antibodies. The labelled proteins were detected by an enhanced chemiluminescence system . Quantification of Western blot bands was performed with ImageJ.33.11H NMR‐based metabolomic profiling to identify and quantify urinary metabolites in 203 IgAN patients with eGFR ≥ 60 mL/min/1.73 m2. With the same criteria applied, MN patients (n = 81), MCD (n = 49), LN (n = 38) patients and healthy controls (n = 146) were profiled for their urine metabolomics. After we excluded the outliers, 201, 77, 47, 36 and 136 patients remained in the IgAN, MN, MCD, LN and healthy control groups, respectively. The baseline clinical characteristics among the study population are shown in Table 2, and the MN and MCD groups had higher urinary random protein/creatinine ratios than the other groups. Within the IgAN groups, 170 (85.4%) patients were on angiotensin‐converting enzyme inhibitors or angiotensin II receptor blockers, and 54 (27.1%) patients were on immunosuppressive drugs.We applied 3.2Unsupervised principal component analysis indicated the composite of metabolites did not clearly separate the study groups in total Figure . When weP < .001), MN (P = .001) and LN (P = .001) patients. The glycine level was higher in the MN cases than in the controls, but the significance did not reach the Bonferroni‐adjusted significance level (P = .017), and the MCD (P = .584) or LN group (P = .798) showed similar levels as the healthy control group.When individual metabolite level was considered Figure , fifteenWhen area under curve of ROC curve by logistic regression model predicting IgAN was calculated, the model including urine glycine level along with clinical variables showed significantly higher area under curve than the base clinical model including age, sex, urine protein‐to‐creatinine ratio, eGFR and mean arterial pressure Table . When th3.3When we tested the correlations between the urinary metabolite levels and the clinical characteristics of the IgAN patients, several positive associations were revealed between the urine protein‐to‐creatinine ratio and several metabolites Figure . Urine gNext, we asked whether certain urinary metabolites were linearly associated with the risk of adverse renal outcomes with panelized spline curves. Several metabolites showed a negative association with the risk of a 30% reduction in eGFR Figure . The assWhen we tested the additive prognostic value of the identified metabolites, the addition of glycine or other metabolites to a clinical model improved the prognostic predictability Table .4 In a3.4Among the metabolites associated with the prognosis of IgAN, glycine was selected as our target urine metabolite for further experimental validation because only glycine was significantly increased in the urine from the IgAN group compared to the disease‐control group. Additionally, this metabolite has been suggested to be associated with a lower risk of incident chronic kidney disease.3.5As glycine is primarily metabolized via the glycine cleavage system, which consists of the T, P, L and H proteins, and via serine hydroxymethyltransferase (SHMT) 1 and 2, we performed immunohistochemistry for these enzymes. The enzymes were mainly detected in kidney tubules Figure . Regardi3.6We found that the expression of protein H of the glycine cleavage system was significantly reduced in the hTECs treated with TNF‐α at 10.0 ng/mL or 20 ng/mL for 1 hour Figure . HoweverThe protein expression of ICAM‐1, pp38 and pp65 was significantly increased in the hTECs treated with 10.0 ng/mL TNF‐α Figure . MoreoveWe next inhibited protein H via the transduction of hTECs with protein H siRNA to explore whether alteration of glycine metabolism through inhibiting the target molecules may replicate the protective effect of high glycine on inflammation in hTECs. Inhibition of protein H by siRNA significantly decreased the endogenous expression of protein H Figure , which a4In this study, we found that glycine levels were increased in the urine of IgAN patients without reduced eGFR compared to those of healthy individuals or patients with MN, MCD, or LN. When assessing the predictive power, measuring urine glycine level showed certain diagnostic value for IgAN. Moreover, higher urinary glycine levels, along with several other metabolites, were significantly associated with the prognosis of early IgAN patients, and the inclusion of urinary glycine levels significantly improved the power of a prognostic prediction model. In the glycine cleavage system, protein H expression was decreased in the tubules of the IgAN patients. Furthermore, we showed that glycine may have a protective role against inflammatory signals by in vitro experiments using hTECs. Inhibition of protein H resulted in an increase in cellular glycine levels along with amelioration of inflammatory signals. Our results suggested that urinary glycine may be a prognostic biomarker for IgAN in those without established kidney function impairment, and glycine metabolism may be a targetable pathway that can improve inflammatory insult in kidney tubules.Although IgAN is defined as a single type of primary glomerulonephritis, the diagnosis mostly depends on morphological changes in kidney pathology, and thus, patients have heterogeneous characteristics and prognosis. Thus, efforts have been made to predict the prognosis of IgAN, and a combination of pathologic grading and clinical characteristics has been used to develop a predictive model with a certain degree of power.in vivo and in vitro experiments.Glycine is a simple amino acid that is present at approximately 170‐330 µmol/L in human or animal blood,in vitro experiment also resulted in amelioration of inflammatory signals in hTECs, glycine may be a targetable metabolite that is associated with prognosis of IgAN.In the current study, we first reported the possibility of a protective role of glycine in the prognosis of early IgAN, which is the most common primary glomerulonephritis worldwide.Our study have several limitations and unanswered questions. First, there were other metabolites, mostly the levels that showed a strong correlation with the levels of glycine, that were associated with the prognosis of IgAN. A future study is warranted to reveal whether they are consequences of altered glycine metabolism or have an independent role in IgAN pathophysiology. Second, although glycine was higher in the IgAN group than in both the healthy control group and the MN/MCD/LN groups, this finding does not mean that the role of glycine is IgAN specific. Given previous reports suggesting various benefits of glycine in ischaemic‐reperfusion injury or inflammatory conditions, the protective role of glycine in the kidney may be due to general mechanisms. This study suggests that glycine can be a prioritized metabolite in early IgAN as the metabolism of glycine was significantly altered in IgAN without prominently reduced eGFR. Third, additional external validation of the results in an independent cohort or a repetitive measurement of urine metabolites during the follow‐up period is necessary to confirm the possible diagnostic or prognostic value of urinary glycine. Despite the relatively large number of IgAN patients, a selection bias remains in this study. Fourth, additional studies are warranted to investigate the possible benefits of interventions targeting glycine in human IgAN. As glycine is an amino acid normally ingested in foods and generally tolerated in humans, additional supplementation with glycine may be considered. However, this study only suggests the possibility but cannot confirm whether increasing the external intake of glycine or targeting the glycine metabolic system can replicate the possible benefits of glycine in human IgAN that was shown in hTECs. Last, our findings may not be applicable to patients with reduced eGFR, as all the study patients had preserved eGFR.In conclusion, urinary glycine levels were increased in IgAN patients without reduced eGFR compared to healthy, MN, MCD, and LN controls. Higher levels of urinary glycine were associated with better prognosis of early IgAN, and the addition of the urine metabolite to the prognostic prediction model of IgAN resulted in improved predictive power. As glycine and related metabolic pathways may have protective roles against inflammatory injury in hTECs, a future study may target glycine when considering interventions to improve the prognosis of IgAN.The authors declare no conflicts of interest.Sehoon Park: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Methodology ; Project administration ; Resources ; Software ; Writing‐original draft (lead); Writing‐review & editing . Jueun Lee: Conceptualization ; Data curation (lead); Formal analysis ; Investigation ; Methodology (lead); Project administration ; Resources ; Software ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Seung Hee Yang: Data curation ; Formal analysis ; Funding acquisition ; Investigation (lead); Methodology (lead); Resources ; Software ; Supervision ; Validation ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Hajeong Lee: Data curation ; Formal analysis ; Investigation ; Resources ; Software ; Supervision ; Writing‐original draft . Joo Young Kim: Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Writing‐original draft . Minkyoung Park: Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration ; Visualization ; Writing‐original draft . Kyu Hong Kim: Data curation ; Formal analysis ; Methodology ; Supervision ; Validation ; Writing‐original draft . Jong Joo Moon: Conceptualization ; Data curation ; Investigation ; Methodology ; Visualization ; Writing‐original draft . Semin Cho: Investigation ; Methodology ; Validation ; Visualization ; Writing‐original draft . Soojin Lee: Data curation ; Formal analysis ; Methodology ; Project administration ; Visualization ; Writing‐original draft . Yaerim Kim: Data curation ; Investigation ; Methodology ; Visualization ; Writing‐review & editing . Hajeong Lee: Data curation ; Funding acquisition ; Investigation ; Project administration ; Supervision ; Validation ; Writing‐review & editing . Jung Pyo Lee: Data curation ; Formal analysis ; Methodology ; Resources ; Software ; Writing‐review & editing . Chang Wook Jeong: Data curation ; Formal analysis ; Investigation ; Methodology ; Supervision ; Writing‐review & editing . Cheol Kwak: Data curation ; Formal analysis ; Methodology ; Supervision ; Writing‐review & editing . Kwon Wook Joo: Conceptualization ; Data curation ; Investigation ; Methodology ; Writing‐review & editing . Chun Soo Lim: Formal analysis ; Investigation ; Methodology ; Software ; Supervision ; Writing‐review & editing . Yon Su Kim: Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Supervision ; Validation ; Visualization ; Writing‐review & editing . Geum‐Sook Hwang: Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Software ; Supervision ; Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing (lead). Dong Ki Kim: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition (lead); Investigation ; Methodology (lead); Project administration ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.