EGFR) gene were among the first EGFR mutations to be identified as oncogenic drivers in non-small cell lung cancer (NSCLC). However, unlike the classical EGFR L858R point mutation or exon 19 deletions, which represent the majority of EGFR mutations in NSCLC, low frequency EGFR exon 20 insertion mutations are associated with de novo resistance to targeted EGFR inhibitors and correlate with a poor patient prognosis. Here, we review the developments over the last 5 years in which pre-clinical studies, including elucidation of the crystal structure of an EGFR exon 20 insertion mutant kinase, have revealed a unique mechanism of kinase activation and steric conformation that define the lack of response of these EGFR mutations to clinically approved EGFR inhibitors. The recent development of several novel small molecule compounds that selectively inhibit EGFR exon 20 insertions holds promise for future therapeutic options that will be effective for patients with this molecular subtype of NSCLC.Inframe insertions of three or more base pairs in exon 20 of the epidermal growth factor receptor ( EGFR) gene are commonly observed in non-small-cell lung cancer (NSCLC). Treatment with EGFR inhibitors has proven successful in many patients harboring such mutations, but patients with EGFR exon 20 insertion mutations do not respond these drugs. Simon Vyse and Paul H Huang at The Institute of Cancer Research in London, UK, review the latest research on the effect that the insertion of three or more nucleotide base pairs into EGFR exon 20 has on the receptor’s structure and activity, which contributes to its poor sensitivity to currently available EGFR inhibitors. Promising preclinical findings with compounds that selectively target the EGFR exon 20 insertion mutant protein may lead to new treatment options for patients with this form of NSCLC.Activating mutations in the epidermal growth factor receptor ( For the adenocarcinoma subtype of NSCLC in particular, significant improvements in progression-free survival (PFS) have been achieved for patients with epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) translocations, ROS1 proto-oncogene receptor tyrosine kinase (ROS1) rearrangements and, B-raf proto-oncogene, serine/threonine kinase (BRAF) mutations due to the effectiveness and availability of therapies that specifically target these molecular drivers.7 Among these advances, the successful use of EGFR inhibitors to treat EGFR mutant positive adenocarcinoma and the subsequent development of third generation inhibitors to tackle acquired drug resistance have become the poster child of targeted therapy in oncology.9Lung cancer is the most common malignant disease and the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) comprising the vast majority (85%) of all lung malignancies.12 In multiple reports, the vast majority of gefitinib-responsive or erlotinib-responsive lung cancers were found to harbor somatic EGFR mutations, while no EGFR mutations could be detected in non-responsive patients.15 These pioneering studies indicated that the presence of EGFR mutations predicted increased sensitivity to EGFR inhibitors, a finding that has subsequently been supported by numerous follow-up studies. The Iressa Survival Evaluation in Lung Cancer (ISEL) clinical trial, for example, compared gefitinib treatment to placebo in NSCLC and found that the response rate (RR) to gefitinib was much higher (37.5%) in patients who possessed EGFR mutations compared to those without (2.6%).16 Another study, the Iressa Pan-Asia Study (IPASS) trial, compared gefitinib treatment to carboplatin plus paclitaxel and showed an extraordinary 72.1% versus 1.1% RR to gefitinib for patients with and without EGFR mutations, respectively, alongside significantly increased PFS in the gefitinib-treated, EGFR mutation-positive group.17 Subsequently, the phase III European Tarceva vs. Chemotherapy (EURTAC) trial also showed a significant advantage for erlotinib  in EGFR mutant patients compared to cisplatin or carboplatin plus either docetaxel or gemcitabine chemotherapy .4A series of studies in the early 2000s observed that distinct epidemiological subgroups of patients with NSCLC  had dramatically enhanced responses to treatment with the ATP-competitive, reversible EGFR inhibitors gefitinib and erlotinib.EGFR activating mutations and represent the vast majority (85–90%) of all observed EGFR kinase domain mutations in NSCLC.18 It is now clear, however, that not all activating EGFR mutations are inherently sensitive to EGFR inhibitors. Inframe base pair insertions in exon 20 also result in constitutive activation of EGFR, but unlike the classical activating EGFR mutations, EGFR exon 20 insertions are associated with de novo resistance to current clinically available EGFR inhibitors.20 Low response rates of between 3–8% for erlotinib, gefitinib and the second generation EGFR inhibitor afatinib have been reported in EGFR exon 20 insertion mutant NSCLC patients  in exon 21 and variable deletions of at least three amino acid residues in exon 19 are together often referred to as ‘classical’ EGFR exon 20 insertion mutations are heterogeneous at the molecular level but can be characterized as inframe insertions or duplications of between 3 and 21 bp (corresponding to 1 to 7 amino acids) clustered between amino acid positions 762 and 774 of the EGFR protein.22 The most common insertion sites in EGFR in NSCLC are shown in Fig. EGFR mutations identified in NSCLC alongside exon 19 deletions and L858R mutations.24 However, reports regarding the incidence and clinical outcome of NSCLC patients with these insertions were initially limited. The frequency of EGFR exon 20 insertions has since been reported as being between 4 and 10% of all observed EGFR mutations in NSCLC.25EGFR exon 20 insertion mutations are largely mutually exclusive with other known oncogenic driver events that are characteristic of NSCLC, such as KRAS mutations,26 and follow similar trends as classical activating EGFR mutations; EGFR exon 20 insertion mutations are enriched in women, non-smokers, Asian populations, and those with adenocarcinoma histology.25EGFR exon 20 insertion mutations have been exclusively reported in NSCLC. Interestingly, however, recent analysis of a rare form of head and neck cancer known as sinonasal squamous cell carcinoma (SNSCC) demonstrated a remarkably high frequency of EGFR mutations (77% of SNSCC tumors), the majority of which were exon 20 insertions .27 This result is striking given that EGFR mutations in head and neck cancer are rare overall, and moreover, the frequency of EGFR mutations is inverse to that of NSCLC; in SNSCC tumors, exon 20 insertion mutations predominate, while exon 19 deletions are detected at low frequency.28 Thus, EGFR exon 20 insertions are not entirely restricted to lung cancer as previously believed but can also act as oncogenic drivers in SNSCC, which represents approximately 3% of all head and neck cancers.30Until recently, HER2, another member of the EGFR family of receptor tyrosine kinases (RTK).31 Although HER2 mutations are present at a much lower frequency (~2% of NSCLC patients) compared with EGFR mutations, exon 20 insertions are, by far, the most dominant type of HER2 aberration in NSCLC, representing greater than 90% of all observed HER2 mutations.33Notably, structurally analogous exon 20 insertion mutations that can promote ligand-independent activation that are also found in 35 For wild-type EGFR, ligand binding is required for activation and induces a conformational change that facilitates receptor homo-dimerization or hetero-dimerization with other EGFR family members. Subsequent autophosphorylation of the tyrosine residues in the intracellular tail of EGFR initiates the formation of large protein complexes that can propagate downstream signaling.38 Many of the signaling pathways activated downstream of EGFR, including the Ras/Raf/Mitogen-activated protein kinase (Ras/MAPK) pathway, phosphatidylinositol 3-kinase/AKT (PI3K/AKT) pathway, and signal transducers and activators of transcription (STAT) pathway orchestrate key cellular processes including cell survival, proliferation, and migration. As such, it follows that dysregulation of EGFR has the potential to contribute to almost all of the classical oncogenic phenotypes that have been described as the ‘hallmarks of cancer’.39EGFR was the first RTK to be identified and belongs to a subset of RTKs known as the EGFR family.42 This shift allows ligand-independent dimerization and activation of the receptor, resulting in constitutive activation of downstream signaling pathways. In lung cancers with classical EGFR mutations, blockade of EGFR signaling with inhibitors can trigger rapid apoptosis in a manner consistent with the ‘oncogene addiction’ model, in which cells are dependent on persistent EGFR signaling for survival.43 Importantly, kinetic analyses have revealed that both L858R and exon 19 deletion EGFR kinases display a greatly reduced affinity for ATP relative to the wild-type receptor.45 Diminished ATP binding has the opposite effect on drug binding; the relative affinity of mutant receptors for reversible ATP-competitive EGFR inhibitors, such as gefitinib or erlotinib, is potently enhanced by alleviating the competitive pressure. This differential sensitivity between wild-type and mutant receptors, combined with the increased dependency of tumor cells on EGFR signaling, provides the wide therapeutic window that makes EGFR inhibitor therapy much more effective in patients with classical activating EGFR mutations.The conformational changes induced by the L858R mutation and exon 19 deletions have been predicted to destabilize the inactive form of EGFR, causing an overall equilibrium shift towards an active over an inactive state.EGFR, the T790M substitution in exon 20, is thought to account for more than half of all cases of acquired resistance to first-generation EGFR inhibitors in NSCLC.46 It was initially predicted that the mechanism of resistance underlying the T790M mutation was steric hindrance imposed by the presence of a bulky methionine residue that would prevent the binding of first-generation EGFR inhibitors to EGFR. However, while the T790M mutation was found to impact the affinity of the mutant EGFR receptor to gefitinib, inhibitor binding was not completely abolished.47 Crucially, further analysis revealed another major factor that contributed to drug resistance: the T790M mutation restored the ATP-binding affinity of the L858R mutant EGFR to almost wild-type receptor levels.47 By increasing the ATP affinity, the T790M mutation diminishes the efficacy of reversible ATP-competitive inhibitors gefitinib and erlotinib, and removes the selectivity that these drugs have for mutant over wild-type EGFR.A second mutational event in 40 Exon 19 deletions in EGFR affect this region via removal of residues from the loop that leads up to the C-helix on the N-terminal side  of EGFR into the drug-binding pocket, resulting in significant steric hindrance.31In 2013, Yasuda et al. solved the first crystal structure of a representative exon 20 insertion EGFR exon 20 insertion mutations are heterogeneous and that the location of the insertion, in particular, may influence the kinetics of drug and ATP binding, ultimately determining resistance or sensitivity to EGFR inhibitors. Indeed, Yasuda et al. also investigated another exon 20 insertion that occurred within the C-helix of EGFR, A763_Y764insFQEA, and unexpectedly found that this mutation had a high affinity for gefitinib in vitro and was highly sensitive to erlotinib in an engineered cell line model.22 Three patients with the A763_Y764insFQEA insertion showed tumor regression or stable disease following erlotinib treatment, leading the authors to conclude that unlike other mutations of its class, this particular exon 20 insertion is sensitive to first-generation EGFR inhibitors. Although its crystal structure was not solved, 3D modeling of the A763_Y764insFQEA insertion suggests that exon 20 insertions that occur before residue 764, within the C-helix itself, might have an activation mechanism and structure that more closely resemble those of the L858R or exon 19 deletion mutant EGFR and therefore would be predicted to be sensitive to first-generation EGFR inhibitors. In agreement, multiple reports have shown partial responses of patients harboring A763_Y764insFQEA insertions to the first-generation inhibitor erlotinib, indicating that these mutations may behave similarly to classical NSCLC EGFR mutations.48 An outstanding question is whether the downstream signaling pathways activated by distinct EGFR exon 20 insertion mutations are the same and to what degree these pathways overlap with those activated by the more common L858R or exon 19 deletion EGFR mutations. Previous work has shown that distinct EGFR mutations drive unique downstream signaling profiles.51 Future characterization of the downstream pathway activation that is unique to EGFR exon 20 insertions may yield mutation-specific therapies that selectively target these pathways.It is important to note that EGFR-mutant positive NSCLC treatment, achieving up to a 72% RR and nearly 10 months PFS.17 By contrast, with the exception of the A763_Y764insFQEA mutation,48 retrospective analysis of clinical data has revealed that first-generation EGFR inhibitors are ineffective in the vast majority of EGFR exon 20 insertion mutant NSCLC patients, with a RR between 8 and 27% and less than 3 months median PFS reported  of neratinib reported a low RR of only 3% in classical EGFR mutant positive lung adenocarcinoma tumors, and there are currently no ongoing clinical trials to assess neratinib in lung cancer.55 An ongoing phase III clinical trial, ARCHER 1050 (NCT01774721), has recently demonstrated significantly longer median PFS after dacomitinib treatment (14.7 months) versus gefitinib treatment (9.2 months) in NSCLC patients harboring classical EGFR mutations, leading to FDA approval for dacomitinib to treat metastatic EGFR mutant-positive NSCLC in September 2018.56 Afatinib is also approved as a first-line therapy for the treatment of NSCLC harboring classical EGFR mutations based on two pivotal studies that demonstrated a significant PFS benefit of greater than 10 months in EGFR-mutant patients.58Three second-generation EGFR inhibitors have been developed: neratinib, dacomitinib, and afatinib. Unlike the first-generation drugs gefitinib or erlotinib, all of these compounds form irreversible, covalent interactions with EGFR at a specific cysteine residue (C797) and have additional activity against at least one other EGFR family member.EGFR mutations pooled from LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6 trials revealed significant clinical activity of afatinib  in rare EGFR point mutations .59 This finding led the FDA to broaden the indication for afatinib to include NSCLC patients who harbor these specific mutations in January 2018. However, in the same analysis, a group of 23 patients harboring EGFR exon 20 insertions treated with afatinib were found to have a minimal response .Currently, there is limited clinical evidence that supports the use of second-generation EGFR inhibitors in exon 20 insertion mutant NSCLC. A recent post-hoc analysis of data from 100 lung cancer patients harboring rare EGFR and HER2 in engineered Ba/F3 and NIH-3T3 cell line models has identified a variable degree of responses to the second-generation inhibitors dacomitinib, neratinib, and afatinib across distinct exon 20 insertions.60 In particular, the authors noted that insertions that introduced a glycine at position 770 were uniquely sensitive to the EGFR/HER2 inhibitor dacomitinib, suggesting a potential therapeutic avenue for patients with mutations that share this feature. In a 2011 phase I clinical trial, out of 6 patients with different EGFR exon 20 insertion mutations, the only patient who demonstrated a partial response to dacomitinib harbored a D770delinsGY mutation, supporting the pre-clinical data.61 However, these mutations are relatively rare—the most common exon 20 insertions lack glycine at position 770; therefore, the majority of patients within this NSCLC molecular subgroup are unlikely to benefit from dacomitinib treatment. Consistent with the clinical data, the pre-clinical study found that only the cell line models expressing a D770delinsGY mutation possessed an in vitro IC50 that was sufficiently low enough (17.5 nM) to predict a response to dacomitinib in patients based on current dosing regimens .62Interestingly, a comprehensive pre-clinical study of exon 20 insertions in 64 Two major T790M-selective irreversible EGFR inhibitors, rociletinib, and osimertinib, have been assessed in the context of EGFR T790M-mutant positive NSCLC, and based on its impressive response rate of greater than 60%, the FDA granted approval for osimertinib as a second-line treatment for EGFR T790M mutant-positive patients following progression on erlotinib or gefitinib in 2017.65 Recently, osimertinib has also demonstrated a significant benefit versus first-generation EGFR inhibitors in previously untreated NSCLC patients harboring classical EGFR mutations. In 556 EGFR-mutant patients, osimertinib-treated patients reached an unprecedented median PFS of 17.2 months versus 8.5 months with first-generation EGFR inhibitor treatment, leading to its recent approval as a first-line therapy.66In response to the identification of the T790M secondary mutation as a major source of resistance to first-generation EGFR inhibitors in NSCLC, third-generation EGFR inhibitors that covalently bind to the C797 cysteine residue of EGFR and maintain selectivity for the double mutant L858R/T790M or exon 19 del/T790M EGFR have been developed.EGFR exon 20 insertions, albeit with a weaker potency than afatinib.67 However, the evidence to support osimertinib as a candidate inhibitor for EGFR exon 20 insertions in vivo remains unclear. A study of lung cancer patient-derived xenograft (PDX) models harboring EGFR exon 20 insertion mutations showed poor responses to the third-generation EGFR inhibitors osimertinib and rociletinib.68 However, Floc’h et al. have recently demonstrated significant antitumor activity of both osimertinib and its circulating metabolite AZ5104 using xenograft models of the H2073 EGFR wild-type lung cancer cell line, which was genetically engineered to introduce either the D770_N771InsSVD or V769_D770InsASV EGFR insertion mutations.69 A phase II clinical trial to assess osimertinib as a treatment for EGFR exon 20 insertion mutant NSCLC (NCT03414814) is ongoing.Pre-clinical in vitro evidence in engineered cell line models has suggested that osimertinib may have some activity against 70 that is currently the most advanced clinical candidate of compounds with the capacity to target EGFR exon 20 insertions. Initially, a phase II study of poziotinib in NSCLC patients with classical EGFR mutations who had acquired resistance to EGFR inhibitors via the T790M mutation or other mechanisms found that poziotinib had minimal clinical activity.71 Poziotinib was subsequently shown to have significant activity against EGFR exon 20 insertions in in vitro models, a result which was mirrored by encouraging clinical data from an ongoing phase II trial (NCT03066206).31 Robichaux et al. used 3D modeling to demonstrate how the rigid C-helix conformation induced by exon 20 insertions results in a relatively small drug binding pocket. This restricted conformation has been predicted to prevent the binding of drugs such as osimertinib, which has a large terminal group and a rigid pyrimidine core, by steric hindrance. By contrast, poziotinib is centered on a less rigid quinazoline core, akin to second-generation EGFR inhibitors. In addition, poziotinib has small terminal and substituent linking groups, making it more compact and flexible compared to current second-generation and third-generation inhibitors. Based on these features, 3D modeling predicts that poziotinib is able to tightly bind the restricted exon 20 insertion binding pocket of EGFR and may also be effective against structurally analogous exon 20 insertions in HER2.Poziotinib (formerly HM781–36B) is a covalent, irreversible inhibitor of EGFR and HER271 However, it is important to note that poziotinib also demonstrated activity against wild-type EGFR in vitro, highlighting a possible narrow therapeutic window that may give rise to dose limitations in EGFR exon 20 insertion patients. Using genetically engineered mouse models harboring EGFR D770insNPG or HER2 A775insYVMA lung tumors, a durable response to poziotinib was shown over 12 weeks of treatment in vivo. Additionally, poziotinib was compared to afatinib in a mouse xenograft model of a patient-derived cell line, YUL-0019, which harbors EGFR N771delinsFH. Although YUL-0019 tumors did not grow in the presence of afatinib, tumor volume remained stable over the course of 10 days. By contrast, poziotinib treatment resulted in a 50% reduction of tumor volume within the same time frame.Following these structural modeling studies, the authors demonstrated potent inhibition of exon 20 insertion EGFR and HER2 mutants in a series of engineered Ba/F3 cell line models. In these cell lines, poziotinib was shown to be uniquely selective for exon 20 insertions over T790M mutants, providing a possible explanation for the initial disappointing results in patients with acquired resistance to EGFR inhibitors in the phase II trial.EGFR exon 20 insertion-positive lung cancer patients (NCT03066206). Although the patient cohort was small, a striking 64% confirmed response rate was achieved in 11 patients harboring EGFR exon 20 insertion mutations. This promising result is in stark contrast to the 8.7% response rate that has previously been observed for afatinib in exon 20 insertion patients.59 It is important to note, however, that the clinical data from this trial are not mature and the median PFS and OS have not yet been reported. Therefore, a question remains: how durable will the response to poziotinib be in patients with exon 20 EGFR insertions? Anticipating the emergence of acquired resistance, Robichaux et al. indirectly showed a possible mechanism that parallels osimertinib resistance by demonstrating that Ba/F3 cells expressing EGFR L858R/T790M/C797S or EGFR exon 19 deletion/T790M/C797S triple mutants are resistant to poziotinib treatment in vitro. The C797S EGFR mutation has been found to be a major clinical mechanism of acquired resistance to osimertinib treatment in patients with classical EGFR mutants who are concurrently T790M positive.72 However, whether the C797S mutation arises in patients with exon 20 insertion mutations has yet to be determined. Similarly, whether poziotinib is clinically effective across a broad spectrum of different EGFR exon 20 insertions with sufficient selectivity over wild-type EGFR to achieve target inhibition with minimal toxicity remains unknown. Based on preliminary results from a phase II clinical trial including data from a small cohort of 30 patients, poziotinib was recently declined Breakthrough Designation Therapy status by the FDA,73 although the trial remains ongoing to assess poziotinib in a larger cohort of NSCLC patients harboring EGFR or HER2 exon 20 insertions (NCT03318939).The authors also reported preliminary data from an ongoing phase II clinical trial for poziotinib in 74 Importantly, Hsp90 inhibitors have shown anti-tumor properties and selectivity for cancer cells over normal cells,75 and although no Hsp90 inhibitors have been approved to date, their potential is being actively explored across many cancer types for which treatment options are limited.76 Recently, pre-clinical data have shown that the EGFR exon 20 insertion mutant kinase associates with the Hsp90 chaperone system and can be degraded through the use of Hsp90 inhibitors.77 A phase II clinical trial of luminespib in EGFR exon 20 insertion NSCLC (NCT01854034) reported that 5 out of 29 (17%) patients achieved partial or complete responses, indicating an improvement over the current RRs to first-generation or second-generation EGFR inhibitors.78 However, both the median PFS (2.9 months) and overall survival (13 months) were short, while the ocular toxicity of this class of drugs remains a concern. Therefore, it is unclear at this stage whether luminespib will have clinical utility in EGFR exon 20 insertion NSCLC patients.Luminespib is an inhibitor of heat shock protein 90 (Hsp90), a molecular chaperone required to maintain stability and assist protein folding for a large number of cellular client proteins, including receptors and signaling components involved in driving oncogenesis.EGFR exon 20 insertions, D770_P772del_insKG and D770>GY, were predicted to increase the electrostatic energy between EGFR monomers and therefore favor the formation of EGFR active dimers.79 This finding led the authors to hypothesize that patients harboring these insertions may be sensitive to a combination of EGFR kinase inhibitors and cetuximab, a monoclonal antibody that binds to the extracellular domain of EGFR and sterically hinders dimer formation.80 Case studies from two patients support the use of cetuximab in combination with EGFR inhibitors or chemotherapy, including one patient (D770>GY) treated with a combination of cetuximab and erlotinib in a phase I clinical trial with a reported PFS of 3.5 years.81 More recently, a clinical study found that three out of four EGFR exon 20 insertion-positive NSCLC patients had partial responses to a combination of afatinib and cetuximab, with a median PFS of 5.4 months.82 These results suggest that the cetuximab and EGFR inhibitor combination may have some efficacy in patients with exon 20 insertions; however, with limited clinical data, further work is necessary to determine the impact of the insertion size and location on the response to cetuximab combinations.Based on in silico structural modeling, two 84 Hypoxia is a common feature of solid tumors and, in particular, has been linked to EGFR inhibitor resistance in pre-clinical models.87 A phase II clinical trial of tarloxotinib in EGFR mutant, T790M-negative NSCLC patients with progression on first-line EGFR inhibitors reported disappointing results; while 7 of 21 patients achieved stable disease, no confirmed partial responses were reported, leading to early termination of the trial.88 However, a recent in vivo study using murine xenografts of two NSCLC cell lines harboring endogenous EGFR exon 20 insertions, CUTO14 (A767_V769dupASV) and CUTO17 (N771_H773dupNPH), has revealed significant tumor regression with tarloxotinib treatment compared with the lack of a tumor response to afatinib.89 These preliminary data suggest that more focused clinical trials are warranted to determine the efficacy of tarloxotinib within the EGFR exon 20 insertion NSCLC patient subgroup.Tarloxotinib is a hypoxia-activated prodrug (HAP) that releases an irreversible EGFR/HER2 inhibitor only under low oxygen conditions.90 Several novel inhibitor compounds have recently been developed and have been shown to directly target the EGFR exon 20 insertion mutant kinase. Although these developments remain at an early stage, published data suggest that these compounds may have significant clinical activity in NSCLC patients harboring exon 20 insertion mutations in EGFR and HER2. In this section, we will discuss the latest pre-clinical evidence that supports the evaluation of these EGFR exon 20 insertion selective inhibitors in lung cancer patients.The development of compounds with selectivity for EGFR exon 20 insertion mutants over wild-type EGFR is crucial to limit the toxicity that arises from wild-type EGFR inhibition in patients.91 Gonzalvez et al. reported selective activity of TAK-788 against 14 different EGFR exon 20 insertion mutant variants expressed in the Ba/F3 cell line model. In the same study, tumor regression was observed in a patient-derived xenograft NSCLC model harboring an EGFR exon 20 insertion mutation following once daily dosing of TAK-788. A phase I/II clinical trial (NCT02716116) is now underway to evaluate the use of TAK-788 in NSCLC patients with exon 20 insertion mutations in EGFR or HER2. A preliminary first report of the dose-escalation cohort demonstrated tolerability of TAK-788 in patients, with an adverse effect profile similar to that of other EGFR inhibitors used in NSCLC.92 Once a maximum tolerated dose is established, the phase II expansion phase aims to determine the overall response rate to TAK-788 in a larger cohort of NSCLC patients harboring EGFR or HER2 exon 20 insertions either with or without brain metastases.TAK-788 (formerly AP32788) is a covalent, irreversible inhibitor that is designed to selectively target the exon 20 insertion mutant forms of both EGFR and HER2 kinases over wild-type EGFR.93 Mass spectrometry analysis demonstrated that TAS6417 forms a covalent interaction with an insertion mutant EGFR kinase at the cysteine residue C797. Further pre-clinical characterization of TAS6417 by Hasako et al. demonstrated selectivity for the D770_N771insNPG mutant EGFR kinase over wild-type EGFR using cell-free in vitro kinase assays. To ensure cellular activity, TAS6417 treatment was shown to inhibit EGFR phosphorylation and cell viability in NIH3T3 cell line models engineered to express a panel of 6 distinct EGFR exon 20 insertion mutations. In addition, as an elegant solution to the paucity of NSCLC cell lines harboring endogenous EGFR exon 20 insertion mutations, the authors employed transcription activator-like effector nuclease (TALEN) mutagenesis to generate a suitable model derived from the H1975 EGFR L858R/T790M mutant NSCLC cell line. TALEN mutagenesis was used to introduce an EGFR D770_N771insSVD insertion into the H1975 cell line and subsequently knock out endogenous L858R and T790M mutations. TAS6417 treatment was then shown to inhibit cell growth of the H1975 EGFR D770_N771insSVD mutant cell line in vitro and in vivo. It was also confirmed that TAS6417 treatment resulted in the inhibition of EGFR phosphorylation in xenografts, coupled with evidence of inhibition of two critical pathways that mediate oncogenic signaling downstream of EGFR, the PI3K/Akt (as measured by pAKT) and Ras/MAPK (as measured by pERK) pathways. Moreover, the level of EGFR signaling inhibition correlated with the plasma concentrations of TAS6417 over a 24 h time course of treatment. Clinical assessment of TAS6417 in NSCLC patients has not yet begun; therefore, it remains to be determined whether the mutant selectivity of the inhibitor is sufficient to achieve a good therapeutic window with low toxicity in EGFR exon 20 insertion-positive patients.TAS6417 (formerly TPC-064) is a newly developed irreversible EGFR inhibitor that was designed to fit into the ATP binding site of the exon 20 insertion EGFR kinase.94 Taking inspiration from EGFR inhibitors that form covalent interactions with EGFR, such as osimertinib and rociletinib,95 compounds were designed using a similar pyrimidine core structure that had the ability to bind the cysteine residue C797 of EGFR. The authors hypothesized that small molecule compounds that can form additional molecular interactions with the active conformation of EGFR would have enhanced potency against the EGFR exon 20 insertion mutant kinase. By examining the crystal structure of wild-type EGFR complexed with osimertinib, Jang et al. observed a deep hydrophobic pocket of EGFR positioned at the back of the ATP-binding site that was not occupied by osimertinib. Based on the original pyrimidine core, a novel series of chemical compound analogues were generated by the addition of substituents that were intended to interact with EGFR in the deep hydrophobic pocket. In particular, the compound identified as ‘1A’ was found to strongly inhibit EGFR phosphorylation in Ba/F3 cell lines expressing EGFR exon 20 insertion mutations and demonstrated less potency against wild-type EGFR compared with the second-generation EGFR inhibitors afatinib and dacomitinib and the EGFR exon 20 targeting compound poziotinib. Jang et al. also showed potent antiproliferative activity against a patient-derived NSCLC cell line, DFCI127, which harbors an EGFR P772_H773insPNP mutation. The poor pharmacokinetic properties of compound 1A, including its low oral bioavailability, short half-life, and high clearance rate, remain to be addressed before assessing the efficacy of this inhibitor in in vivo models.Jang et al. have recently employed a structure-guided rational drug design strategy to develop a currently unnamed lead compound, referred to as ‘compound 1A’, which has activity against EGFR and HER2 exon 20 insertion mutant kinases.EGFR exon 20 insertion mutations.19 Crystal structures and in vitro analyses of exon 20 insertion EGFR mutant kinases have revealed that, unlike L858R or exon 19 deletion mutant EGFR, the majority of exon 20 insertions do not possess diminished ATP binding or enhanced affinity for first-generation EGFR inhibitors over wild-type EGFR, thereby reducing the therapeutic window required for their clinical benefit.22 Compounds with the ability to overcome these issues and target EGFR exon 20 insertion mutations, therefore, present an exciting opportunity for clinical use in this patient population. For example, the most advanced clinical compound to date, poziotinib, has achieved a 64% objective response rate, a significant improvement upon the previous 8.7% objective response rate for afatinib59 and 17% objective response rate for the Hsp90 inhibitor luminespib.96 Several outstanding challenges lie ahead–for instance, given the significant molecular heterogeneity of the size and location of distinct EGFR exon 20 insertions, it will be important to determine whether all patients within this molecular subtype of NSCLC will universally respond to these inhibitors. Moreover, the long-term clinical durability of EGFR exon 20 insertion-selective drugs remains unclear, and identifying eventual resistance mechanisms and strategies to overcome them will be crucial in achieving durable patient responses. Considerable efforts are underway to explore the concept of synthetic lethality to identify additional dependencies in classical mutant EGFR-driven models that can be targeted to overcome drug resistance.97 An unresolved question is whether EGFR exon 20 insertion-driven NSCLC will share similar downstream signaling profiles and vulnerabilities that can be exploited therapeutically. Nevertheless, based on encouraging preliminary data with EGFR exon 20 selective compounds, it is anticipated that one or more of these inhibitors will achieve good tolerability and clinical efficacy in patients that will lead to regulatory approval. Moving forward, there is the exciting prospect that these selective kinase inhibitors will provide novel and effective treatment options for patients with EGFR exon 20 insertion mutant-positive NSCLC in the near future.To date, clinically approved targeted EGFR inhibitors have failed to effectively treat NSCLC driven by


Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer that lacks expression of the estrogen and progesterone receptor and does not overex-press human epidermal growth factor 2 receptor protein. TNBC is associated with special characteristics, including aggressiveness, poor prognosis, and treatment response. Non-invasive blood-based molecular markers such as cell-free DNA (cfDNA) variables have been shown to be putative markers in breast cancer prognosis.β-actin gene with different lengths (99 and 394 bp) was used to evaluate the integrity index 394/99.The cfDNA quantity and integrity were assessed in a case-control study of 96 breast cancer patients including 46 triple negative and 50 non-triple negative compared with 50 unaffected controls. A quantitative real-time PCR approach based on the quantification of two amplicons of the Both cfDNA integrity index and quality were significantly elevated in breast cancer patients but integrity index can be considered as the more reliable diagnostic marker. The statistically significant increase of cfDNA quantity and integrity was observed in TNBC patients, somehow associated with nodal metastasis (p<0.001).Elevated cfDNA concentration and integrity index in breast cancer patients compared with normal control and significant difference observed between TNBC and non-TNBC may be considered as a possible effective non-invasive diagnostic and prognostic molecular marker in breast cancer. The Progesterone Receptor (PR), Estrogen Receptor (ER), and Human Epidermal growth factor Receptor 2 (HER2) are well known predictive markers in breast cancer. The Triple-Negative Breast Cancer (TNBC) is classified as a group with no expression of ER, PR, and HER2, accounting for 15–26% of all breast cancer patients. TN breast cancer cases usually show poor prognosis and treatment response via gene expression microarray analysis.The terms “triple negative” and “basal-like” are not completely synonymous Circulating cell-free DNA (cfDNA) as tumor-derived fragmented extracellular DNA provides a non-invasive, personalized genomic snapshot of a patients' tumor and has huge potential in prenatal diagnosis The main source of cfDNA in healthy subjects is apoptotic cells but, the cancer cells release longer DNA fragments resulting from necrosis, autophagy, or mitotic catastrophe The ratio of cancer cell-derived DNA and normal cells-derived DNA called DNA integrity index was found to be increased in cancer patients and may be considered as a malignancy indicator The association between plasma cfDNA concentration and TNBC has not been fully elucidated. The aim of the present study was to investigate the plasma cfDNA concentration and integrity index in breast cancer patients focusing on triple negatives compared with non-triple negative counterparts and normal control.The peripheral blood of 96 patients [46 Triple Negative (TN) and 50 non-TN breast cancer patients] from Imam-Khomeini Hospital  and 50 unaffected female blood donors were taken. The patient inclusion criteria were the histopathological diagnosis of ductal carcinoma and availability of Immunohisto-Chemistry (IHC) results for HER-2, ER, PR status and other pathologic diagnostic information . ReceiviThe study was approved by the Ethical Committee of the National Institute of Genetic Engineering and Biotechnology (NIGEB) based on the Helsinki declaration. All individuals signed an informed consent to participate in the study.ml in ethylene-diamine-tetraacetic acid) was obtained and the plasma was separated by two sequential centrifugations at 1000 g, 4°C. The plasma DNA was extracted using the QIAmp DNA Blood Midi Kit  according to the manufacturer’s instructions.Peripheral blood , 1.0 μl each primer (0.4 mM), 2 μl water, and 6 μl of extracted DNA. The thermal cycling conditions comprised cycles at 95°C for 10 min and 40 cycles at 95°C for 10 s and at 60°C for 60 s followed by cycles at 72°C for 60 s in duplicate. Sample DNA concentration was extrapolated from the standard. The OVCAR3 cell line was used as a control, and a negative no template control was included in each run. DNA integrity index was calculated as the ratio between the 394 and 99 bp amplicons of the β-actin gene. The primer sequences were as follows:Quantification and qualification of plasma DNA were assessed by quantitative real-time PCR (QRTPCR) by ABI 7500/7500 fast real-time system  using human bp): 5′AGGATCTTCATGAGGAGTCAGTCAG-3′, Reverse primer (β-actin 394 bp): 5′-TTAGCTTCCACAGCACAGCC-3′.Common forward primer: 5′-CCACACTGTGCCCATCTACG-3′, Reverse primer (β-actin 99 Data were assessed with SPSS (Version 16) software using Mann Whitney and Kruskal Wallis tests. Results were expressed as means±standard deviation, and the p-value less than 0.05 was considered statistically significant. The predictive capability (diagnostic performance) of each biomarker was investigated by means of the area under the ROC (Receiver-Operating Characteristics) curve (AUC).The cfDNA concentration and integrity index were both significantly elevated in breast cancer patients compared with control (p<0.001). The ROC curve analysis confirmed its possible diagnostic effect .The data revealed the significant increase of plasma cfDNA concentration and integrity index in cancer patients both in TNBC and non-TNBC compared with normal control (p<0.001) , Table 2The Lymph Node (LN) positive groups, as well as stage IV, showed significantly higher plasma cfDNA concentration  and inteThe cfDNA levels in plasma/serum seem to be an important universal malignancy marker besides therapy response biomarker in several tumor entities bp, whereas cfDNA from the necrotic origin is of higher molecular weight It was previously reported that apoptotic cfDNA is fragmented into 180–200 β-actin gene amplification was significantly higher in both breast cancer groups including triple negative and non-triple negatives compared with normal control. The mean of plasma cfDNA concentration was significantly higher in triple negative breast cancer group compared with non-triple negative counterparts. This higher concentration was associated with higher cancer stages and lymph node involvement. The highest level of plasma cfDNA concentration was observed at stage IV and LN-positive patients. The quantification of two amplicons of the β-actin gene with different lengths (99 and 394 bp) was used to evaluate the integrity index 394/99. The integrity index was elevated in TNBC compared with non-TNBC as well as lymph node positive patients but there were no statistically significant differences in cfDNA integrity index among different cancer stages. Being triple negative, involving the lymph nodes and having higher stages of the disease, all are the signs of poor prognosis and invasive characteristics of tumors. It could be concluded that higher plasma cfDNA concentration and integrity were associated with more invasive characteristics in cancer. Our data was somehow in line with a study in Egyptian breast cancer patients that reported the higher percentage of cfDNA as well as long cfDNA fragments in breast cancer patients than controls and correlated with higher cfDNA concentration and integrity with HER2 positivity, metastasis and non-treatment response et al provided another explanation of cfDNA fragmentation The blood cfDNA concentration has been reported as the potential screening and diagnostic biomarker in various cancers such as breast The high cfDNA concentration in cancer patients compared with normal individuals may be due to the reason that in cancerous tissues in spite of normal physiologic condition, most of the released DNA from apoptotic and necrotic cells are not removed by macrophages efficiently In conclusion, the elevated cfDNA concentration and integrity index in breast cancer patients was compared with normal control and significant difference observed between TNBC and non-TNBC may be considered as a possible effective non-invasive prognostic and diagnostic molecular marker in breast cancer.


ARMS2, rs10737680 in CFH, and marginally significant difference for rs800292 in CFH. Between CSC without irregular PED and PCV, rs10490924, rs10737680, and rs800292 were significantly different. In contrast, CSC with irregular PED and CNV revealed no SNP showing significant difference. Between CSC with irregular PED and PCV, only rs10490924 was significantly different. CSC with irregular PED and CSC without irregular PED revealed significant difference for rs800292, and marginal difference for rs10737680. These findings suggest CSC patients with irregular PED are genetically different from those without irregular PED and may have genetic and pathophysiologic overlap with AMD patients.We evaluated phenotype and genotype correlation of central serous chorioretinopathy (CSC) patients with or without irregular pigment epithelial detachment (PED) on optical coherence tomography (OCT). For CSC, a flat, irregular protrusion of retinal pigment epithelium (RPE) with hyper-reflective sub-RPE fluid on OCT was defined as an irregular PED. Participants were classified into 5 subgroups; (1) total CSC (n = 280) (2) CSC with irregular PED (n = 126) (3) CSC without irregular PED (n = 154) (4) typical choroidal neovascularization (CNV) (n = 203) and (5) polypoidal choroidal vasculopathy (PCV) (n = 135). Ten known major AMD-associated single-nucleotide polymorphisms (SNPs) were analyzed. Age, sex adjusted logistic regression was performed for the association between subgroups. Association analysis between CSC without irregular PED and CNV revealed that significant difference for rs10490924 in  Central serous chorioretinopathy (CSC) is a generally self-limiting disease characterized by localized serous detachment of central retina with retinal pigment epithelial abnormality. Recent advance of imaging devices like optical coherence tomography (OCT) showed serous retinal detachment frequently combined with retinal pigment epithelial detachment (PED) with underlying thickened choroid and choroidal hyperpermeability. CSC is also known as one of the pachychoroid spectrum disease due to its thick choroid.2. Pachychoroid spectrum diseases refer to a group of clinical entities that have a common feature of pachychoroid and share an underlying disease mechanism. Pachychoroid spectrum diseases include pachychoroid pigment epitheliopathy (PPE), CSC, pachychoroid neovasculopathy (PNV), and polypoidal choroidal vasculopathy (PCV)3. In this spectrum, PNV has been postulated as a possible precursor of PCV4.Pachychoroid features include focal or diffuse increase in choroidal thickness, choroidal hyperpermeability, and dilated choroidal vessels8. We also performed genotypic comparison study on the patients with CSC, PCV and control. However, we could not find any genetic association between CSC and PCV9.If these diseases are in the same spectrum of disease, they might share similar genetic architecture. Previously, some report showed CSC is related with age-related macular degeneration (AMD) risk genotype but the direction is apposite10. With progress of optical coherence tomographic imaging, PED in CSC showed diverse size, shape and nature like small RPE bump like elevation, large dome shape or regular PED and sometimes flat, irregular PED.PED is a well-known feature of CSC. It occurs in 70–100% of cases with CSC17. Due to diverse features of OCT, OCTA, and genetic studies, we presumed that CSC is not a single disease but may be mixed disease of similar phenotype. Because of the mixed nature of the disease, CSC is vaguely classified as acute, chronic or atypical CSC without established concrete definition. This is also a good reason why CSC has diverse clinical features and different visual prognosis.Some of CSC patients with irregular PED showed vascular de-correlation signal by OCT angiography (OCTA) which suggests hidden sessile choroidal neovascularization (CNV) present in the irregular PED. There have been some studies revealing that irregular PED is associated with the presence of CNV in eyes with chronic CSCConsidering previous studies on the association of the irregular PED in CSC and the CNV or pachychoroid spectrum disease, irregular PED in CSC can be regarded as a hidden CNV in CSC or early stage of PNV.Consequently, we speculate that not the whole CSC patients, but CSC patients with irregular shaped PED may have similar genetic risk factors those associated with PCV or neovascular AMD. There has been no study on genetic difference between CSC patients with irregular PED and those without irregular PED. To find out clinical implications of irregular PED in CSC, we investigate whether CSC patients with irregular PED have different genotype compared to those without irregular PED and also analyze the genetic association between the CSC patients with irregular PED and the patients with PCV or neovascular AMD.P < 0.001). The proportion of the female in group of CSC with irregular PED was higher than group of CSC without irregular PED . The mean age (±standard deviation [SD]) was 48.1 (±8.1) years in total CSC, 72.3 (±8.4) years in CNV, and 67.7 (±7.5) years in PCV (P < 0.001). The mean age was higher in group of irregular PED (+) than irregular PED (−) in CSC . The mean subfoveal choroidal thickness (SFCT) (±SD) was 397.9 (±97.7) μm in total CSC, 217.1 (±76.6) μm in CNV, and 269.0 (±94.6) μm in PCV (P < 0.001). The mean SFCT was slightly thinner in group of irregular PED (+) than irregular PED (−) in CSC .The demographic details of the study population are shown in Table CFH was significantly different between CSC with irregular PED and CSC without irregular PED . Rs10737680 in CFH showed marginally significant difference between the two groups  , rs10737680 in CFH  and marginally significant difference for rs800292 in CFH  and typical CNV (n = 203), rs10490924 in 80 and tyARMS2-HTRA1; P = 4.17 * 10−3, OR = 0.41; Supplementary Table In the association analysis between CSC with irregular PED (n = 126) and typical CNV n = 203), there was no SNP showing significant difference  and typical CNV (n = 203), rs10490924 in  and typiThe summary of the above analyses was presented in Table CFH were significantly different, and the rs10737680 in CFH showed marginally significant difference. These findings suggest not CSC patients but CSC patients with phenotype of irregular PED may have genetic and pathophysiologic overlap with neovascular AMD patients.Based on the age, sex adjusted association analysis with 10 AMD-associated SNPs, patients without irregular PED showed significant difference with CNV patients in 2 SNPs, marginally significant difference in 1 SNP, and significant difference with PCV patients in 3 SNPs. In contrast, patients with irregular PED revealed no SNP showing significant difference with CNV patients, and only one SNP showing significant difference with PCV patients. In the association analysis between patients with irregular PED and those without irregular PED, the rs800292 in 14. One study using OCTA showed that CNV was detected more in chronic CSC eyes with irregular PED  than in those with regular PED 11. Given the fact that CSC is included in the pachychoroid spectrum disease, another study using OCTA was similar to the previous one and it revealed that type 1 neovascular tissue was visualized in most of the study eyes  with pachychoroid spectrum diagnoses and shallow irregular PED14. In the study of Dansingani KK et al.18, pachychoroid with neovascularization revealed only one SNP showing significant difference with neovascular AMD and 4 SNPs  showing significant difference with pachychoroid without neovascularization among 12 AMD-associated SNPs that were analyzed. Pachychoroid without neovascularization revealed 7 SNPs showing significant difference with neovascular AMD in their study. Although the study was different from our study because we excluded CSC patients with any evidence of choroidal neovascularization, it was similar to the present study in that two subgroups of pachychoroid disease were genetically different and one subgroup was relatively closer to the neovascular AMD.The age, sex adjusted association analysis revealed that patients with irregular PED were genetically different from patients without irregular PED. In regards to genetic profile, patients with irregular PED were closer to patients with CNV or PCV than those without irregular PED. These findings suggest CSC patients with irregular PED may have genetic overlap with neovascular AMD patients. The findings imply that patients with irregular PED may have more tendency to have hidden CNV or to develop into CNV or PCV. This implication is similar to the previous studies that showed irregular PED in CSC or pachychoroid disease appears to be a risk factor of CNV4. The current finding that CSC patients with irregular PED may have genetic overlap with neovascular AMD patients implies irregular PED in CSC might be the prodromal change for neovasularization. There have been some studies on genetic overlap between CSC and AMD. De Jong et al. showed that one SNP in ARMS2 and three SNPs in CFH were significantly associated with chronic CSC among 19 AMD-associated SNPs that were analyzed5. Recent study revealed that the G allele of rs800292 and T allele of rs10490924 are significantly associated with the CNV development in patients with CSC19. Considering the fact that CSC with irregular PED is more likely to have hidden CNV, the result of the study corresponds with the findings of the present study which showed the frequency of the G allele of rs800292 or T allele of rs10490924 was higher in CSC with irregular PED than that in CSC without irregular PED  was lower and the frequency of the CFH rs800292 minor allele  was higher in PNV patients.Considering that CSC with irregular PED is more likely to develop into neovascular change, findings in the current study are in line with the previous studies that implied CSC resides within a spectrum of diseases with a pachychoroid-driven process including PPE, CSC, PNV, and PCV25 and the mean age is relatively younger, ranging 39–51 years26, the above demographic findings suggest that patients with irregular PED have different phenotype from the typical CSC patients than those without irregular PED have.In addition to the genetic profile, patients with irregular PED were demographically different from patients without irregular PED. The proportion of female and the mean age of the patients were relatively higher in patients with irregular PED than those without irregular PED. Given the fact that CSC is more prevalent in menARMS2 , rs10737680 in CFH , rs800292 in CFH ). Similarly, association analysis between CSC without irregular PED and typical CNV, rs10490924 in ARMS2, rs10737680 in CFH were significantly different . Rs800292 in CFH showed marginally significant difference between the two groups . Although some of the p-values did not reach the significant difference, the directions of the ORs were similar for the comparison between total CSC and typical CNV (or PCV) . Overall, the association of CFH  with total CSC or CSC without irregular PED was risky and the association of ARMS2 (rs10490924) was protective, compared to typical CNV or PCV. The minor allele of the rs800292 was known to be protective for exudative AMD in Korean27 and Chinese28. This minor allele was also known to be risky for CSC in Japanese20 and Western European5 population. These findings can explain the risky association of the minor allele of the rs800292 for CSC compared to typical CNV or PCV in the current study. The minor allele of the rs10737680 was known to be protective for AMD in East Asian29 and Chinese30. There has been no previous study analyzing the association of rs10737680 with CSC. Hence, the risky association of the minor allele of the rs10737680 for CSC compared to typical CNV or PCV in the current study cannot be directly explained. The observation that some SNPs of the CFH have opposite effects in CSC compared to AMD was also revealed in the previous studies20, but the exact rationale and the mechanism are unclear. CFH is an inhibitor of the alternative complement pathway of the complement system. CFH also binds and interacts with adrenomedullin which has been shown to induce vasodilation of choroidal vascular beds, affect choroidal blood flow, and increase microvascular permeability34. The exact mechanisms for risk association of some SNPs in CFH with CSC are not clear, but the interaction of CFH with adrenomedullin may be the possible explanation of the association of CFH with CSC in that the main pathogenesis of the CSC is related with the choroidal vasodilation and hyperpermeability. In our study, the minor alleles of the rs800292 and rs10737680 were risky for only CSC without irregular PED, not for CSC with irregular PED, compared to typical CNV or PCV. Further studies on the exact mechanisms for the risky association of some SNPs in CFH only with CSC without irregular PED (and not with CSC with irregular PED) are warranted. The minor allele of the rs10490924 was known to be risky for exudative AMD in Korean27 and for AMD in East Asian29. This minor allele was also reported to be protective for CSC in Western European5. These findings can explain the protective association of the minor allele of the rs10490924 for CSC compared to typical CNV or PCV in the current study. A recent study revealed that ARMS2 interacts with components of the extracellular matrix (ECM)35. Disturbances in the ECM at the level of RPE and/or choroid could affect the susceptibility to cellular detachments, which may be the possible mechanisms for the protective association of ARMS2 with CSC5.Association analysis between CSC without irregular PED and PCV revealed that significant difference for 3 SNPs  of Seoul National University Bundang Hospital  and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects before participation in the study.Patients with CSC, exudative AMD with typical CNV, and PCV were recruited at SNUBH retina clinic from January 2009 to January 2016. All patients underwent comprehensive ophthalmologic evaluation, including measurement of best-corrected visual acuity, slit-lamp biomicroscopy, indirect fundus exam, fluorescein angiography (FA), indocyanine green angiography , and optical coherence tomography .38. Patients receiving PDT before the first spectral-domain (SD) OCT at our institute or patients receiving exogenous corticosteroids were excluded. Patients showing evidence of diabetic retinopathy, retinal vascular diseases, or other diseases that can cause macular exudation, such as exudative age-related macular degeneration (AMD), PCV were excluded in the CSC patients.The CSC group was defined as a group of patients with following characteristics: serous subretinal fluid on optical coherence tomography, focal leakage spot (ink blot) or smokestack pattern or ≥1 area of multifocal diffuse leakage, or presence of irregular retinal pigment epithelium window defects on fluorescein angiography and corresponding hyperfluorescence on indocyanine green angiography40, we analyzed the typical CNV and PCV patients separately. Cases having evidence of retinal–retinal or retinal–choroidal anastomosis on ICGA were defined as retinal angiomatous proliferation (RAP). Patients with RAP having choroidal neovascular membrane were included in the typical CNV group. The PCV group was defined as hyperfluorescent polypoidal choroidal vasculature with branching vascular network on ICGA with concomitant exudation or hemorrhage41.The typical CNV group was determined by demonstration of choroidal neovascular membrane on FA without any evidence of PCV on ICGA. According to the recent studies suggesting PCV belongs to pachychoroid spectrum diseases42, a dome-shaped protrusion of RPE with sub-RPE fluid was defined as a ‘regular PED’. A flat, irregular non-dome-shaped protrusion of RPE with ‘at least partially hyper-reflective’ sub-RPE fluid was defined as an’irregular PED’. According to the optical density of the sub-RPE fluid of flat PED, there were also categories of flat PED with ‘hypo-reflective’ sub-RPE fluid and flat PED with ‘indeterminate optical density’  Small irregular protrusion of RPE without sub-RPE fluid was defined as a ‘RPE bump’. Using high speed mode with the spectralis OCT, we checked the 30° * 20° field of view (8.7 mm * 5.8 mm) obtained from the 25 serial B scan to classify the PED. Representative OCT images of regular PED, flat PEDs, and RPE bump were shown in Fig. Based on the OCT findings in patients with CSCFinally, study participants were classified into five subgroups; (1) total CSC  (2) CSC with ‘irregular PED’ (3) CSC without ‘irregular PED’ (4) typical CNV and (5) PCV.http://genome.sph.umich.edu/wiki/Exome_Chip_Design). Genotype calling was carried out using Illumina’s GenTrain version 2.0 clustering algorithm in Genome Studio software (V2011.1). Cluster boundaries were determined using Illumina’s standard cluster file. After visual inspection on SNPs with high missing rates, completely missing SNPs  were removed for further analysis. In total, 646 samples [CSC (n = 281), CNV (n = 221), PCV (n = 144) were successfully genotyped with average call rate >99%.DNA was extracted from whole blood by DNA extraction kit . Study samples processed on the HumanExome BeadChip  includes 243,345 markers focused on protein-altering variants. Details about SNP content and selection strategies can be found at the exome array design webpage (see http://pngu.mgh.harvard.edu/purcell/plink). Among the genotyped SNPs, twelve major AMD-associated SNPs including 9 SNPs replicated in the previous studies of East Asian29, and 2 SNPs replicated in the previous studies of Korean27 were analyzed to investigate the difference of genetic profile and association with AMD among subgroups. The SNP data was cleaned by removing SNPs with a low genotyping pass rate (greater than 5% of genotypes missing from the entire cohort), SNPs with a low minor allele frequency (less than 0.2% of MAF), and SNPs in those subjects without disease that were not in Hardy Weinberg Equilibrium (HWE p < 10−6). After quality control, among 12 major AMD-associated SNPs, 10 SNPs  remained for further analysis. The remained 10 SNPs were rs800292, rs1061170, rs10737680 in CFH, rs6795735 in ADAMTS9, rs4698775 in CFI, rs429608 in C2-CFB, rs943080 in VEGFA, rs334353 in TGFBR1, rs10490924 in ARMS2-HTRA1, and rs3764261 in CETP. After quality control, 280 patients with CSC, 203 patients with CNV, and 135 patients with PCV remained for the statistical analysis.Demographics of the study population were compared among subgroups using the t-test, χ2 test and the one-way analysis of variance (ANOVA). Genotype data cleaning and analysis was performed using PLINK (k = 0.916). When discrepancies arose for the OCT-based phenotyping, the two observers discussed their evaluations and came to an agreement for final phenotype.The OCT-based phenotyping for CSC was performed independently by two observers (S.C.C and N-K.R); 1) regular PED, 2) flat PED with ‘at least partially hyper-reflective’ sub-RPE fluid , 3) flat PED with ‘hypo-reflective’ sub-RPE fluid, 4) flat PED with ‘indeterminate optical density’, 5) RPE bump, and 6) only SRF. All measurements were performed based on 1:1 micron images. The inter-observer agreement of the phenotyping was evaluated with Cohen’s kappa  between CSC with ‘irregular PED’ and CSC without ‘irregular PED’; (2) between total CSC with and typical CNV (or PCV); (3) between CSC with ‘irregular PED’ and typical CNV (or PCV); and (4) between CSC without ‘irregular PED’ and typical CNV (or PCV). For the SNP analysis, the minor allele, or less frequent allele, for each SNP was tested for association with disease of the case for each pair of the subgroups mentioned above using the chi square test in PLINK. Logistic regression was also performed to investigate the age, sex adjusted association of the genotype with disease of the case. A Bonferroni correction was performed, and the individual test for the 10 SNPs were considered significant at P < 0.005, corrected for 10 tests. The p-value of the age, sex adjusted analysis using logistic regression was finally used to determine the significant association.supplementary information.


Renal denervation (RDN) is a catheter-based ablation procedure designed to treat resistant hypertension (RH). The objective of our study is to determine the effect of RDN on blood pressure and renal function in patients with RH in comparison to medical therapy alone. We performed an extensive literature search for randomized control trials (RCT) reporting office and 24 hr. blood pressure changes and estimated glomerular filtration rate (eGFR) at baseline and 6 months. We calculated a weighted standardized mean difference of blood pressure and renal outcomes between RDN and control groups using random effects models. Our search yielded 608 studies of which we included 15 studies for the final analysis. A total of 857 patients were treated with RDN and 616 patients treated with medical therapy ± sham procedure. Only 5 studies were double-blinded RCT with sham control. The adjusted standardized mean difference in the change in office based systolic and diastolic pressures ; 24 hr. systolic and diastolic pressures ; and eGFR (p = 0.20) from baseline to 6 months is statistically insignificant with significant heterogeneity. Subgroup analysis showed that among sham controlled trials, 24 hr. systolic blood pressure showed a modest but statistically significant benefit favoring renal denervation in patients with RH. Our meta-analysis of 15 RCTs showed no significant benefit of RDN on blood pressure control in patients with resistant hypertension. Subgroup analysis of sham control studies showed a modest benefit in 24 hr. systolic blood pressure at 6 months with RDN. The prevalence of RH based on recent studies is ranging from as low as 12.8%2, while others have shown RH to be up to 25–30% in the general hypertensive population3. A 2012 study by Dougherty et al. showed the incidence of resistant hypertension to be 1.9% within 1.5 years of newly diagnosed hypertensive patients undergoing treatment4.Resistant hypertension (RH) is defined as blood pressure that remains above guideline-directed goal despite the concurrent use of at least three antihypertensive agents of different classes, one of which is a diuretic5. Since then, there have been numerous studies and randomized control trials to evaluate the effectiveness of this procedure in treating RH with variable results.When hypertension remains uncontrolled with lifestyle changes and antihypertensive medications, renal denervation has been a proposed intervention to aid in the treatment of RH since it was first used a decade agoet al. did not show any significant effect on blood pressure in patients with RH following renal denervation6. Over the past year, multiple randomized control trials evaluating the effect of renal denervation on RH showed promising results including: WAVE-IV7, SPYRAL HTN-ON MED8, INSPiRED9, and a Polish study by Warchol-Celinska et al.10 We, therefore, conducted a systematic review and meta-analysis based on randomized control trials to assess the efficacy of renal denervation in patients with RH at 6 months in comparison to medical therapy alone ± sham procedure.One of the most recent meta-analysis w by Fadl Elmula 13, while the remaining 10 trials were open-label clinical trials with a control group treated with optimal medical therapy21. Radiofrequency (RF) ablation was performed in all trials except the WAVE-IV7, where ultrasound energy based (US) ablation was performed. Only 13 studies reported a mean change in office based systolic and diastolic blood pressures, and 14 studies reported a mean change in 24 hr. systolic and diastolic blood pressure between baseline and 6 months post-randomization. Only 11 studies reported a change in estimated glomerular filtration rate (eGFR) between baseline and 6 months post-randomization in the treatment and control groups. The baseline characteristics of the included studies are included in Table et al.10 which was a low-quality study  at 6 months in the RDN group was −1.7 ± 11.75 vs −2.39 ± 11.67 in the control group.The change in blood pressure (24 hr. and office) at 6 months and renal function in patients in the renal denervation (RDN) and the control group for each study is listed in Supplementary Table 2 = 87.5%). The results are summarized in Fig. The adjusted standardized mean difference in the change in systolic blood pressure (office) at 6 months between RDN and control groups was −0.24  −0.60–0.11, p = 0.18) which is statistically insignificant. Subgroup analysis showed no statistical significance in both the sham controlled (p = 0.81) and non-sham controlled studies (p = 0.20). There was a high degree of heterogeneity noted among the studies (I2 = 85.8%). The results are summarized in Fig. The adjusted standardized mean difference in the change in systolic blood pressure (24 hr.) at 6 months between RDN and control groups was −0.22 , which was statistically insignificant. However, subgroup analysis among sham control studies showed statistical significance favoring renal denervation (p = 0.02). A high degree of heterogeneity was noted among the studies included in the main analysis (I2 = 92.3%), which likely accounted for the lack of difference noted between both groups. The results are summarized in Fig. The adjusted standardized mean difference in the change in diastolic blood pressure (office) at 6 months between RDN and control groups was −0.34  which was not statistically significant. Subgroup analysis showed no difference in both the sham controlled (p = 0.52) and non-sham controlled studies (p = 0.16). There was a high degree of heterogeneity noted among the studies (I2 = 79.6%). The sham control studies showed no heterogeneity (I2 = 0%), however, the result remained unchanged. The results are summarized in Fig. The adjusted standardized mean difference in the change in diastolic blood pressure (24 hr.) at 6 months between RDN and control groups was −0.19  which was not statistically significant. Subgroup analysis showed no statistical significance in both the sham controlled (p = 0.10) and non-sham controlled studies (p = 0.18). There was a high degree of heterogeneity noted among the studies (I2 = 32.9%). The results are summarized in Fig. The adjusted standardized mean difference in the change in eGFR at 6 months between RDN and control groups was 0.11  which was statistically insignificant. Subgroup analysis showed no statistical significance in both the sham controlled (p = 0.69) and non-sham controlled studies (p = 0.07). There was a low degree of heterogeneity noted among the studies  was performed in 2010, operators have become more proficient with the procedure leading to better outcomes. This also explains the heterogeneity of the results among the trials. With the exception of the change in 24 hr. systolic blood pressure, subgroup analysis (Sham vs No sham) and meta-regression analysis did not change the significance of blood pressure and renal function outcomes between RDN and control groups. The discrepancies observed between changes in 24 hr. blood pressure and office blood pressure between the two groups could be explained by “regression to the mean” effect, which occurs as a result of using specified office blood pressure thresholds while enrolling patients in these trials25. Sham control studies allow accounting for potential confounding factors including “regression to the mean effect”, the placebo effect of the procedure and improved medication adherence after renal denervation.Significant heterogeneity was observed when all the included studies were analyzed together. The differences in blood pressure changes could in part be explained by differences in study design, type of control group and type of RDN procedure performed. It is widely believed that the experience of an operator plays a major role in the success of the procedure. In the initial trials, patients were enrolled from centers with very low operator volume which probably affected the success of the procedure. However over a period of time, since the first trial  Placebo effect in patients who underwent sham controlled procedures. (2) Improvement in adherence to medications as all the patients in the trials was monitored using medication adherence tools such as witnessed medication intake, maintenance of diary, assessment of plasma drug concentration, etc. and (3) Recruiting patients who weren’t maximized on medical therapy.8 trials showed a significant reduction in blood pressure with RDN. The use of multiple catheter systems could potentially contribute to the heterogeneity noted in our analysis.The Symplicity Catheter system  is commonly used in contemporary practice. However it has several limitations including (1) presence of single electrode leading to longer ablation time, (2) presence of unipolar electrode makes a selection of ablation site challenging and (3) inability to ablate deep renal sympathetic nerve due to low radiofrequency power of the catheter limiting the penetration depth. To overcome these limitations, Symplicity Spyral catheter system  and EnligHTN catheter system  were introduced. They are multi-electrode catheters containing 4 electrodes and can simultaneously ablate 4 locations at the same time. Results from SPYRAL HTN-OFF MED and SPYRAL HTN-ON MED26. The significance was predominantly driven by SPYRAL-HTN-ON MED trial8. The renal denervation procedure (using RF ablation) performed in this trial targeted branch vessels beyond the proximal main renal artery which was unique. Animal studies have shown a greater reduction in renal norepinephrine levels with RF ablation if the distal extra-renal artery branches were targeted27. This hypothesis was further tested by Petrov et al. in a non-randomized study (N = 119) which compared the efficacy of blood pressure reduction comparing conventional renal artery denervation strategy to a novel renal denervation strategy that targets the distal branches of the renal artery in addition to the proximal renal artery. They showed a greater decrease in office and 24 hr. systolic and diastolic blood pressure among the patients who underwent the novel procedure compared to the conventional procedure. This technique of radiofrequency RDN may explain the predominantly greater reduction in blood pressure noted in the RDN group in the SPYRAL-HTN-ON MED trial.The adjusted pooled analysis of the 5 sham controlled studies showed a modest but significant benefit in the mean difference in 24 hr. systolic blood pressure change at 6 months which is contrary to the prior meta-analysis published28 was performed to test head to head comparison between US ablation, conventional RF ablation of main renal arteries and RF ablation of main + side branches of renal arteries. The study showed that significantly higher reduction in ambulatory blood pressure with US ablation compared to conventional RF ablation of main renal arteries. However, no difference in blood pressure reduction was noted between patients who underwent US ablation compared to patients who underwent RF ablation of main and side branches of renal arteries. This further explains the degree of heterogeneity noted in our analysis.RADIOSOUND-HTN trial17 or plasma drug concentration20 for a total of 344 patients . The remaining 11 studies assessed medication adherence using compliance diary or validated questionnaires. Medication non-adherence is noted among 50% of hypertensive patients and its prevalence increases especially in patients with RH. Accurate assessment of medication non-adherence is difficult given the invasive nature of the tests and incorporation into routine practice remains a challenge given the tests are cost-intensive.A predominant confounder in randomized control trials evaluating the efficacy of a novel therapy is the medication adherence among study participants. In this instance, if the medication adherence significantly improves after the patient undergoes renal denervation; the true effect of the procedure would be difficult to estimate. Among the 15 included studies, only four studies used robust methods to assess medication adherence using witnessed medication intake29. The positive results noted in the newer trials did not affect the overall result of the meta-analysis.We have also shown that patients who underwent RDN had no significant changes in renal function (eGFR) compared to the medical therapy group at 6 months post-renal denervation, supporting the safety profile of the procedure. Our findings are consistent with prior published studiesThe meta-analysis reported here combines data across studies in order to estimate treatment effects with more precision than is possible in a single study. Limitations include incorporating studies with different control arms and designs to increase the study population and maximizing the likelihood of estimating a treatment effect. However, the results of the subgroup analyses would be considered more meaningful given the lower heterogeneity. This specifically applies to the pooled analysis of 5 studies which showed a modest improvement in 24 systolic blood pressures with renal denervation. Publication bias might account for some of the effects we observed. Smaller trials are, in general, analyzed with less methodological rigor than larger studies, and an asymmetrical funnel plot suggests that selective reporting may have led to an overestimation of effect sizes in small trials. The lack of individual participant level data for meta-analysis and subgroup analysis is a limitation of our study. Given the nature of meta-analysis, the inherent weakness of the individual studies will be inherited in our study. The variability of expertise among all the physicians performing renal denervation could potentially affect the outcome of the procedure, thereby the overall outcome of the individual trials.An extensive electronic search of Embase, Scopus, Web of Science, PubMed, and manual review of the literature was performed for relevant articles using the following search terms: “renal denervation”, “renal sympathetic denervation”, and “resistant hypertension”. The search was restricted to publications in English and the final search was performed till from Jan 2008 to June 2018 (Supplementary data).30.Studies were included if they satisfied the following criteria: (1) Age of the study population >18 yrs.; (2) Original randomized control trials comparing renal denervation with medical therapy ± sham procedure, (3) Published a detailed study protocol, (4) Follow-up duration of ≥6months, (5) Patient population diagnosed with resistant hypertension on ≥3 blood pressure medications, (6) Reported estimated glomerular filtration rate (eGFR), average office or ambulatory systolic and diastolic blood pressure at baseline and 6 months following intervention, and (7) Reported characteristics of the overall study population. Studies were excluded in case of partial/complete overlap of the study population with a study already included in the analysis and also in cases of incomplete or incorrect reporting of data. All eligible studies complied with Consolidated Standards of Reporting Trials (CONSORT) quality criteria31. When both intention to treat and per protocol outcomes were reported by the authors, we only considered intention to treat values for our analysis. We directly contacted the authors when additional information was missing from the initial studies.The initial search was performed by two reviewers , and studies were selected for inclusion by mutual consensus. In case of disagreement, a third reviewer (RA) resolved disagreements through discussion to achieve a consensus. Once the studies were selected for inclusion, the data were screened to meet the inclusion criteria and thereafter the following data points were extracted: number of anti-hypertensive medications, class of anti-hypertensive medications used, eGFR, average office or ambulatory systolic and diastolic blood pressure at baseline and 6 months following the intervention. Additionally, cumulative demographic and baseline characteristics of the patient population included in the study were also extracted. The quality of included studies was assessed using the Jadad scale for randomized control trials2 statistic value greater than 50%. Publication bias was visually assessed using Deek’s funnel plot followed by a trim-and-fill procedure. The Deek’s funnel plot utilizes regression of diagnostic log odd’s ratio against 1/square root (effective sample size) and further weighting by effective sample size. A p-value of the slope coefficient of <0.10 indicated asymmetry and thereby publication bias. The significance of asymmetry in the funnel plot will be assessed using Eggers test and a trim-and-fill test will be performed if applicable to correct asymmetry33. We performed a sensitivity analysis by type of control arm (sham vs no sham controlled).Continuous variables are presented as mean and standard deviation and categorical data as percentages. The aim of our analysis was to assess the change in blood pressure (24 hr. and office measured) and renal function in patients who underwent renal denervation in comparison to patients on optimal medical therapy. We calculated an unadjusted pooled change in eGFR, office and 24 hr. blood pressure in both renal denervation and control groups. We calculated a weighted standardized mean difference among outcomes between the renal denervation group and control group with random effects models (DerSimonian and Laird). Statistical heterogeneity was defined as I34. All the above analyses were performed in R, version 3.4.3, using “mada” package.Meta-regression analysis was performed to identify potential sources of heterogeneity. We performed meta-regression for covariates including sample size (less or more than 100), region of study, year of publication, quality of study, sham vs no sham controlled, age, gender, body mass index, proportion of patients with hypertension, coronary artery disease and diabetes mellitus to identify potential sources of heterogeneity. This meta-analysis was performed in compliance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelinesOur meta-analysis of 15 randomized control trials showed no significant benefit of renal denervation on blood pressure control in patients with resistant hypertension. Subgroup analysis of sham control studies showed a modest benefit in 24 hr. systolic blood pressure at 6 months with renal denervation. Our analysis substantiates the safety profile of the procedure o based on renal function at 6 months post procedure. Given the evidence of procedure-related factors, objective assessment of drug adherence, novel renal denervation techniques, and sham control study design affects the overall study outcomes; future clinical trials must account for these variances in order to better assess the effectiveness of renal denervation in patients with RH. Furthermore, the type of ablation therapy used and target sites for ablation  must be explored in future trials. Based on the current evidence, patients with resistant hypertension and no identifiable secondary cause , maximized on lifestyle interventions and medical therapy by a hypertension specialist may benefit from renal denervation with an experienced operator.Supplementary Data


CREBBP and EP300, contributed to low BMD following a literature review. A novel biologic pathway identified in this study highlighted a gene module and several genes as playing possible roles in the pathogenesis of glucocorticoid- induced derangement in BMD.Derangement in bone mineral density (BMD) caused by glucocorticoid is well-known. The present study aimed to find key biological pathways associated with low BMD after glucocorticoid treatment in asthmatics using gene expression profiles of peripheral blood cells. We utilized immortalized B cells (IBCs) from 32 childhood asthmatics after multiple oral glucocorticoid bursts and peripheral blood mononuclear cells (PBMCs) from 17 adult asthmatics after a long-term use of oral glucocorticoid. We searched co-expressed gene modules significantly related with the BMD Z score in childhood asthmatics and tested if these gene modules were preserved and significantly associated with the BMD Z score in adult asthmatics as well. We identified a gene module composed of 199 genes significantly associated with low BMD in both childhood and adult asthmatics. The structure of this module was preserved across gene expression profiles. We found that the cellular metabolic pathway was significantly enriched in this module. Among 18 hub genes in this module, we postulated that 2 genes,  We previously reported that polymorphisms located in CRHR1, TBCD and RAPGEF5 were associated with low BMD in childhood asthmatics after a long-term treatment of glucocorticoids5. However, so far, there has been no comprehensive study to elucidate biologic mechanisms underlying glucocorticoid-induced derangement in BMD in asthmatics who are frequently treated by glucocorticoid.Derangement in bone mineral density (BMD) caused by glucocorticoids is well-known. Glucocorticoid treatment is the most common cause of secondary osteoporosis7. The present study aimed to find key biological pathways associated with low BMD after glucocorticoid treatment in patients with asthma using weighted gene co-expression network analysis (WGCNA).A network based analysis is an emerging and promising approach and provides new insights into BMD genetics by grouping genes into modules which are enriched for particular biological processes8. Moreover, recent reports showed that PBMCs could be used to assess glucocorticoid sensitivity or efficacy of osteoporosis treatment10. Taken together, we may use gene expression profiles of peripheral blood cells for the purpose of searching what biologic pathways are related to glucocorticoid-induced derangement in BMD.For this purpose, we used gene expression profiles of immortalized B cells (IBCs) from childhood asthmatics after multiple oral glucocorticoid bursts and those of peripheral blood mononuclear cells (PBMCs) from adult asthmatics after a long-term use of oral glucocorticoids. Peripheral blood is an easily accessible tissue, although it is not directly involved in bone biology. However, it was suggested that molecular profiling of peripheral blood might reflect physiological and pathological events occurring in different tissues of the body2 > 0.8, Figure A total of 32 childhood asthmatics whose IBCs and BMD profiles were available and 17 adult asthmatics enrolled  were taken as hub genes due to their high connectivity and association with the BMD Z score . Replication analysis was done in two ways: preservation of module (the consistency of network module structure across gene expression profiles) and association with the target phenotype . Among 10 modules identified in the discovery cohort, three modules  were significantly preserved in the replication cohort Fig. . Module e Figure .Figure 3in vitro perturbation of modules identified in gene expressions of Sham-treated IBCs by Dex, we measured changes of the module structure between Sham- and Dex-treated IBCs. Among the 10 modules, only the magenta module identified in Sham-treated IBCs was not preserved in Dex-treated IBCs . The most significantly enriched pathway was the cellular metabolic pathway . This pathway consists of 10,751 genes and 150 genes out of 199 genes belong to the BMD Z score-associated common module overlapped. Enriched pathways identified are shown in Table 4. We performed a pathway analysis using genes mapped to 200 SNPs and identify the cellular macromolecular metabolic process (GO:0044260) as the most significant candidate causal pathway .To assign biological meaning to interpretability of the BMD Z score-associated common module, we performed pathway enrichment analyses in GO biological process categories with its membership genes. To increase the specificity in the enrichment results, we set a more stringent threshold of overlap (intersection of query and test sets ≥ 3) and excluded consideration of inferred electronic annotations , immune response (GO:0006955), response to hypoxia (GO:0001666), and response to extracellular stimulus (GO:0009991) which were similar with GO pathways identified in this study. The cellular metabolic pathway representing the chemical reactions and pathways by which individual cells transform chemical substances never been recognized in previous studies focused on osteoporosis. To get a further insight, we performed a pathway analysis using the genes mapped to top-200 SNPs that were significantly associated with the bone mineral accretion in childhood asthmatics in our previous genome-wide association study4. We identified the cellular macromolecular metabolic process (GO:0044260) as the most important candidate causal pathway. This pathway is a sub-pathway of the cellular metabolic pathway (GO:0044237)12. These SNPs could not be directly linked to gene expression/modules identified in the present study, as genomics data were generated from different populations. However, we thought that this result would be an additional evidence suggesting a possible role of the cellular metabolic pathway in osteoporosis pathogenesis. Cellular metabolism is an important part of the bone biology and homeostasis14, but, so far, no direct evidence connecting this pathway to glucocorticoid-induced BMD derangement has been issued, although the cellular amine metabolic pathway (GO:0033240), a sub-pathway of the cellular metabolic pathway, was reported to be enriched in gene expressions of PBMC from patients with osteoporosis compared to normal control15. Further studies are needed to confirm our observations.Membership genes in this module were significantly overrepresented in the cellular metabolic pathway (GO:0044237). A previous report exploring differential gene expression profiles on PBMC by the graph clustering approach and GO term enrichment analysis showed 9 gene clusters associated with osteoporosis12). For this reason, we selected highly connected intramodular18 hub genes. By reviewing the literatures, we selected two genes of putative interest: EP300 encoding E1A binding protein p300 and CREBBP coding CREB binding protein (CBP). CBP and p300 are highly related transcriptional co‐activators possessing histone acetyltransferase activity and are known to participate in the activities of hundreds of different transcription factors involved in a variety of cell functions16. Up-regulation of miR-132-3p could inhibit osteoblast differentiation in part by decreasing p300 expression17, and CBP could mediate osteoblast-adipocyte lineage plasticity by interaction with Maf19. In addition, both CBP and p300 were known to be important co-factors mediating transforming growth factor-beta and bone morphogenic protein signaling which have fundamental roles in bone homeostasis20, and to modulate parathyroid hormone regulation of osteoblast21. Interestingly, it has been also reported that these two proteins are involved in steroid receptor signaling22. Steroid receptors require coactivators for efficient activation of target gene expression and these coactivators might contribute to tissue specific responses to steroid hormone. Likewise, CBP and p300 might function additively or antagonistically to each other depending on their relative concentrations and type of target tissue, to influence the sensitivity of tissues to glucocorticoids23. In addition, it was reported that CREBBP mutations resulted in impaired expression of GC receptor responsive gene and impaired histone acetylation and transcriptional regulation25. Given that CBP and p300 are involved not only in bone homeostasis but also in glucocorticoid receptor signaling, a gene module identified in this study which contained CREBBP and EP300 may play an important role in glucocorticoid-induced derangement in BMD.It was not easy to give biological meanings to our findings at the pathway level, as identified pathways were diverse and contained many genes . Using this dataset, we could find that the magenta module (the BMD Z score-associated common module) identified in this study was preserved in gene expressions of Sham-treated osteoblasts , and different ways of exposure to glucocorticoid (short-term burst vs. chronic use) were considered before generalizing our findings. Previously, age-dependent alterations in osteoblast and osteoclast activity were reported40. In general, short-term glucocorticoid use was associated with mild adverse effects, whereas long-term use might be associated with more serious sequel including osteoporosis41. For this reason, we provided information on the hub genes and enriched biologic pathways of genes belong to the blue module, a childhood asthmatics-specific gene module, in the supplementary information n Figure . CurrentIn conclusion, the potentially significant genes and pathways identified in our analysis may help further elucidate of the molecular mechanisms and provide novel insights regarding glucocorticoid-induced derangement in BMD in asthmatics.All experimental protocols were approved by the Institutional Review Board of the corresponding institutions. This study was approved by the Institutional Review Board of the Brigham and Women’s Hospital (2002-P-00331/41) and the Seoul National University Hospital (H-1508-095-695). Informed consents were obtained from all study participants and a parent and/or legal guardian, if subjects were under 18. And all methods were carried out in accordance with relevant guidelines and regulations. The overall study design is presented in Fig. 42. In asthma exacerbations, short courses of oral prednisone were prescribed per protocol to them. Each burst consisted of 2 mg/kg per day (up to 60 mg) of prednisone for 2 days followed by 1 mg/kg per day (up to 30 mg) for 2 days. Subsequent maintenance of prednisone was allowed when the improvement was not sufficient. BMD measurements of the lumbar spine (L1-L4) were performed annualy during the study period by means of dual-energy x-ray absorptiometry [Hologic  or Lunar ]. The BMD Z score that represents a difference between the measured bone density in a subject and the average bone density in age- and sex-matched controls was calculated by using the CAMP internal ref. 2. In the present study, the final BMD Z score measured after mean follow-up duration of 4.3 years was used as a target phenotype.The discovery cohort consisted of Non-Hispanic white children from the Childhood asthma management program (CAMP) trial whose IBCs were available43. At the enrollment, BMD was measured at the lumbar spine (L1-L4) by dual-energy X-ray absorptiometry [Lunar ]. Like childhood asthmatics, the BMD Z score was used as a target phenotype.The replication cohort was drawn from adult asthmatics who were treated at Seoul National University Hospital, Seoul, Korea. All of them received more than 15 mg of prednisone per day continuously for at least one year before enrollment for the control of their symptoms. Doses above 10 mg of prednisone per day result in significant bone loss44 IBCs derived from childhood asthmatics from the CAMP trial were cultured in RPMI 1640 medium (Sham-treated IBCs) and treated with dexamethasone (10−6 M) for 6 hours (Dex-treated IBCs). Dex-treated IBCs were used to measure in vitro perturbation of gene expressions by Dex. PBMCs from adult asthmatics were cultured in RPMI 1640 medium for 6 hours (Sham-treated PBMCs). Gene expression levels were measured using the Illumina HumanRef8 v2 BeadChip  for childhood asthmatics and the Affymetrix GeneChip Human Gene 2.0 ST  for adult asthmatics. We removed probes with bad chromosome annotation and probes in X or Y chromosome. We then did variance stabilizing transformation and quantile normalization respectively to reduce the effects of technical noises and to make the distribution of expression level for each array closer to normal distribution.As previously describedwww.r-project.org). We performed WGCNA on each gene expression profile with the R package “WGCNA”45. To allow comparability of data from different microarray platforms, we first calculated the mean expression value of each gene in two expression profiles (Sham-treated IBCs and Sham-treated PBMCs) after collapsing probes by genes. We then compared the mean expression level of genes of them and selected top-5000 genes in common with the highest correlation. Using 5,000 genes we first characterized unsigned correlation networks and their relationships with each other and found gene modules in gene expression profiles from childhood asthmatics. A module was defined as a group of highly interconnected genes45. We computed eigengene values of modules identified and performed multivariate linear regression analysis adjusted by baseline age, gender, BMD Z score, vitamin D level, Tanner stage, and body mass index Z score. Eigengene values can be effective and biologically meaningful tools for studying the relationships between modules of a gene co-expression network and phenotypes46. The BMD Z score-associated module was defined when its eigengene value was significantly associated with the BMD Z score, a target phenotype. We next tested if the BMD Z score-associated modules identified from childhood asthmatics and were preserved in gene expression profiles from adult asthmatics using the R package “NetRep”47. NetRep can quantify the preservation of gene co-expression modules across different datasets and produce unbiased p values based on a permutation approach to score module preservation without assuming data are normally distributed47. The BMD Z score-associated common module denoted a module which was identified in gene expression profiles from childhood asthmatics and significantly preserved in gene expression profiles from adult asthmatics. We then calculated an eigengene value (the first principle component) of the BMD Z score-associated common module in adult asthmatics and performed multivariate linear regression analysis adjusted by age, gender, vitamin D level, body mass index, and smoking status.Analysis was performed with R version 3.4.3 (48. g:Profiler (https://biit.cs.ut.ee/gprofiler/) provides an adjusted P value calculated in a manner that accounts for the hierarchical relationships among the tested gene sets. Finally, we searched the key driving hub genes in the BMD Z score-associated common modules, as an occurrence of certain disease is less likely associated with the abnormality of the whole genes involved in the biological pathway. Hub genes were defined by module connectivity, measured by absolute value of the Pearson’s correlation (cor.geneModuleMembership > 0.8) and clinical trait relationship, measured by absolute value of the Pearson’s correlation (cor.geneTraitSignificance >0.2)49. For the pathway analysis of SNPs, we selected the top-200 SNPs (Table S) from our previous report4 and put those SNPs into ICSNPathway (http://icsnpathway.psych.ac.cn/). The ICSNPathway enables us to identify candidate causal SNPs and pathways from genome-wide association study by one analytical framework50. Parameters used were as follow: specify SNP, P value < 1.0E-5; linkage disequilibrium cutoff, r2 = 0.8; Rule of mapping SNPs to genes, within gene; Population, CEU; Pathway gene set size, minimum = 5 and maximum = 100; FDR cutoff, 0.05.We then performed Gene ontology (GO) pathway overrepresentation analyses of the BMD Z score-associated common modules using “g:Profiler” (database version: r1730_e88_eg35)Supplementary Information.