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This file contains Supplemental figures, tables, and legends that accompany the main figures, as indicated in each legend. There are 3 supplemental tables and 6 supplemental figures. Table S1. List of potential target of the GNS compounds that were profiled in Figure 1 of the main text. Table S2. Half-maximal inhibitor concentration of the EGFR inhibitors tested. Table S3. Blood-brain-barrier penetration of the GNS compounds as assessed by the indicated pharmacokinetic measurements. Figure S1. Structural modeling studies show the binding characteristics of the indicated EGFR inhibitors. Figure S2. The effects of each EGFR inhibitor in the cell lines expressing mutant or wild type EGFR. Figure S3. In vivo effects of the GNS compounds. Figure S4. In vivo anti-tumor effects of the GNS compounds. Figure S5. In vivo anti-tumor effects of the GNS compounds in intracranial EGFR-mutant lung cancer. Figure S6. In vivo anti-tumor effects of the indicated EGFR inhibitors in intracranial EGFR-mutant lung cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200–11. ©2017 AACR.

Background:The response of 1st-line EGFR-TKI treatment in advanced EGFR-mutated lung adenocarcinoma patients are highly effective, but its sustainability and clinical course are quite variable from patient to patient. In this study, we investigated the clinical impact of concomitant genetic mutations analyzed by targeted NGS on PFS and acquired resistance in advanced EGFR-mutated lung adenocarcinoma patients. Methods: Eighty-five advanced NSCLC patients harboring EGFR mutations were enrolled prospectively in multi-centers from 2019 to 2022 (NCT04122833). We performed the targeted next generation sequencing on 324 cancer-related genes by Foundation One CDx with pre-treated tumor samples. First- or second-generation EGFR-TKIs(gefitinib, afatinib, or erlotinib) were administered in 1st-line setting. After the progression, tissue re-biopsy or plasma liquid biopsy (FoundationOne Liquid CDx) for NGS if tissue biopsy is difficult or risk was performed. Results: Of the 85 patients (70.6% of female, 65.8% of nonsmoker, 56.4% of E19del and 38.8% of E21 L858R mutation), 50 patients experienced a disease progression in November, 2022. The median PFS was 20 months (95 %CI: 15.2-24.8). The most frequent co-mutations were TP53 (47.1%), CDKN2A/B loss (34.1%), MTAP loss (20%), NKX2-1amp(15.3%), MDM2amp (14.1%), RMB10, CCNE/CCND1 amp, NFKB1 amp (11.8%) and CDK4/6 amp (10.6%). Patients with TP53, CDK4/6 amp and MYC amp were independently associated with shorter PFS. In a multivariate analysis, tumors with copy number alterations such as CDK4/6 amp or Myc amp were also independently associated with shorter PFS. However, the CDKN2A/2B loss, MTAP loss, and MDM amp were not related with the PFS. In the number of co-mutations, patients harboring ≥5 co-mutations identified by NGS had shorter median PFS than patients with 0-1 or 2-4 co-mutations. (mPFS 0-1: 2-4: ≥5 co-mutations=35: 18: 9.3 months, p Conclusion: We have demonstrated that concomitant mutations detected by targeted NGS analysis provide significant impact on the drug response and clinical course of advanced EGFR-mutated adenocarcinoma patients treated by 1st-line EGFR-TKIs. It is suggested that targeted NGS along with PCR-based detection will be necessary for precision medicine-based individualized practice of 1st-line EGFR-TKI-based combination treatment. Citation Format: In Ae Kim, Seung Joon Kim, Sung Yong Lee, Chang Min Choi, Jae Cheol Lee, Tae Won Jang, Seung Hun Jang, Chan Kwon Park, Wan Seop Kim, Jae Young Hur, Hee Joung Kim, Young Whan Kim, Key Young Lee. Analysis of concomitant genetic alterations in advanced EGFR-mutated lung adenocarcinoma by targeted NGS: A multicenter prospective and real world study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3886.

Background Accumulating evidences indicate that AXL overexpression or activation is associated with cancer progression and acquired resistance to targeted anti-cancer drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Despite recent development of several drugs that target multiple receptor tyrosine kinases (RTKs), drugs that selectively target AXL signaling are extremely rare. Short nucleic acid aptamers are non-immunogenic molecules with high binding affinity and specificity to their target molecules that could potentially be used as a novel cancer treatment. Methods Modified-DNA aptamers were selected on the basis of its ability to bind recombinant human AXL. AXL aptamers were selected for their inhibition of AXL and then selected aptamers were tested for their use to overcome acquired resistant to EGFR-TKI on a lung cancer cell with acquired resistance to erlotinib. Results These new AXL aptamers inhibited cell viability to an extent of 30–40% in HCC827/ER cells with acquired resistance to erlotinib. The possible mechanism of overcoming the acquired resistance may be by inhibiting the activation of Akt and Erk. Although, aptamers effectively decreased cell viability of erlotinib‐resistant cell line, the combination of aptamers and erlotinib did not synergistically decrease the survival of the resistant cell line. Conclusions We developed newly modified DNA aptamers that selectively bind to AXL receptors, and assessed their efficacy in a human lung cancer cell with acquired resistance to EGFR-TKI.

BACKGROUND: Extracellular vesicles (EVs) are membrane-bound and nanometer-sized particles released from most types of cells, containing double-stranded DNA reflecting mutational status of the parental tumor cells. Furthermore, epidermal growth factor receptor (EGFR) genotyping using EV-derived DNA (EV DNA) in bronchoalveolar lavage fluid (BALF) showed almost 100% sensitivity in patients with advanced non-small cell lung cancer (NSCLC). METHODS: We assessed the technical performance of DNA derived from BALF-EV (BALF EV DNA) in targeted next-generation sequencing (NGS) for detection and quantification of mutations compared with the matching tissue DNA in 20 lung adenocarcinomas. RESULTS: DNA yields, tumor purity, and depth of coverage were higher using the tissue DNA than using the BALF EV DNA. However, estimated library size was not significantly different between the two samples, and BALF EV DNA yielded longer fragments than tissue DNA. Overall mutation concordance between the two samples were 56% for nonsynonymous somatic mutations and increased to 81% for clinically significant mutations. By-variant sensitivity for clinically significant somatic mutations increased from 62% to 83% in the NGS of BALF EV DNA. Allele frequencies of EGFR and TP53 were higher in tissue DNA (10–25%) than in BALF EV DNA (

Background Despite surgical resection, early lung adenocarcinoma has a recurrence rate of 20–50%. No clear predictive markers for recurrence of early lung adenocarcinoma are available. Targeted next-generation sequencing (NGS) is rarely used to identify recurrence-related genes. We aimed to identify genetic alterations that can predict recurrence, by comparing the molecular profiles of patient groups with and without recurrence. Methods Tissues from 230 patients with resected stage I–II lung adenocarcinoma (median follow-up: 49 months) were analyzed via targeted NGS for 207 cancer-related genes. The recurrence-free survival according to the number and type of mutation was estimated using the Kaplan–Meier method. Independent predictive biomarkers related to recurrence were identified using the Cox proportional hazards model. Results Recurrence was observed in 64 patients (27.8%). In multivariate analysis adjusted for age, sex, smoking history, stage, surgical mode, and visceral pleural invasion, the CTNNB1 mutation and fusion genes (ALK, ROS1, RET) were negative prognostic factors for recurrence in early-stage lung adenocarcinoma (HR 4.47, p = 0.001; HR 2.73, p = 0.009). EGFR mutation was a favorable factor (HR 0.51, p = 0.016), but the CTNNB1/EGFR co-mutations were negative predictors (HR 19.2, p p = 0.02). Conclusions: Targeted NGS can provide valuable information to predict recurrence and identify patients at high recurrence risk, facilitating selection of the treatment strategy among close monitoring and adjuvant-targeted therapy. Larger datasets are required to validate these findings.

With advances in target therapy, molecular analysis of tumors is routinely required for treatment decisions in patients with advanced non-small cell lung cancer (NSCLC). Liquid biopsy refers to the sampling and analysis of circulating cell-free tumor DNA (ctDNA) in various body fluids, primarily blood. Because the technique is minimally invasive, liquid biopsies are the future in cancer management. Epidermal growth factor receptor (EGFR) ctDNA tests have been performed in routine clinical practice in advanced NSCLC patients to guide tyrosine kinase inhibitor treatment. In the near future, liquid biopsy will be a crucial prognostic, predictive, and diagnostic method in NSCLC. Here we present the current status and future perspectives of liquid biopsy in NSCLC.

Background/Aims We performed a large-scale, retrospective, nationwide, cohort study to investigate the risk factors for lung cancer among never-smoking Korean females. Methods The study data were collected from a general health examination and questionnaire survey of eligible populations conducted between January 1, 2003 and December 31, 2004; the data were acquired from the tailored big data distribution service of the National Health Insurance Service. After a 1-year clearance period, 5,860,922 of 6,318,878 never-smoking female participants with no previous history of lung cancer were investigated. After a median follow-up of 11.4 years, 43,473 (0.74%) participants were defined as “newly diagnosed lung cancer”. Results After adjusting for all variables at baseline, the variables older age, lower body mass index (BMI), less exercise, frequent alcohol drinking, meat-based diet, rural residence, and previous history of cancer were associated with a higher incidence of lung cancer. Low BMI (< 18.5 kg/m2: hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.27 to 1.40) was a significant independent risk factor; as BMI decreased, HR increased. Negative associations between BMI and lung-cancer development were also observed after controlling for age (p for trend < 0.001). Drinking alcohol one to two times a week (HR, 1.25; 95% CI, 1.21 to 1.28) and eating a meat-based diet (HR, 1.08; 95% CI, 1.01 to 1.15) were associated with lung-cancer incidence. Conclusions Modifiable baseline characteristics, such as BMI, exercise, alcohol consumption, and diet, are risk factors for lung-cancer development among never- smoking females. Thus, lifestyle modifications may help prevent lung cancer.

Near-threshold computing (NTC) has recently emerged and been considered as a strong candidate for future energy-efficient computing. However, adverse impacts from process variation such as delay and power fluctuations within die as well as across dies are much more severe than the super-threshold regime. In particular, static random access memory (SRAM)-based components (e.g., cache memories) are easily affected by process variation in NTC, resulting in large delay fluctuations. It incurs a huge loss in the maximum clock frequencies of processors, which eventually leads to huge yield losses. In this paper, we first analyze L1 data cache yield in NTC and reveal an inefficiency of frequency binning for yield improvement in NTC. We then introduce a variable latency L1 data cache for NTC to obtain a sufficient yield. By allowing the higher cache access cycles, we can improve cache yield with only a little performance overhead. Moreover, we propose an adaptive line migration technique which improves performance and energy efficiency of variable latency caches. The cache line which is expected to be frequently accessed in the near future is dynamically migrated to the fastest way in a cache set. According to our evaluation, our cache architecture greatly improves cache yield with only a little performance, energy, and area overhead.

Background Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site. Methods Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)-mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping. Results The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P

Targeted NGS, widely applied to identify driver oncogenes in advanced lung adenocarcinoma, may also be applied to resected early stage cancers. We investigated resected EGFR-mutated lung adenocarcinoma mutation profiles to evaluate prognostic impacts. Tissues from 131 patients who had complete resection of stage I–IIIA EGFR-mutated lung adenocarcinoma were analyzed by targeted NGS for 207 cancer-related genes. Recurrence free survival (RFS) was estimated according to genetic alterations using the Kaplan–Meier method and Cox proportional regression analysis. The relapse rate was 25.2% (33/131). Five-year RFS of stages IA, IB, II, and IIIA were 82%, 75%, 35%, and 0%, respectively (p &lt, 0.001). RFS decreased with the number of co-mutations (p = 0.025). Among co-mutations, the CTNNB1 mutation was associated with short RFS in a multivariate analysis (hazard ratio: 5.4, 95% confidence interval: 2.1–14.4, p = 0.001). TP53 mutations were associated with short RFS in stage IB–IIIA (p = 0.01). RFS was shorter with EGFR exon 19 deletion (19-del) than with mutation 21-L858R in stage IB–IIIA tumors (p = 0.008). Among 19-del subtypes, pL747_P753delinS (6/56, 8.9%) had shorter RFS than pE746_A750del (39/56, 69.6%), the most frequent subtype (p = 0.004).

Simple Summary We reviewed characterization and the role of DNAs derived from extracellular vesicles focusing on its use for identifying biomarkers. Extracellular vesicles contain double-stranded genomic DNA reflecting the mutational status and methylation profile of the parental tumor cells. Many studies demonstrated higher stability, sensitivity, and specificity of extracellular vesicle DNAs in comparison to cell-free DNAs, demonstrating a high potential for clinical application as a source for liquid biopsy. Moreover, the horizontally transfer ability of extracellular vesicle DNAs could be utilized in therapeutics. Abstract Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential.

The conventional linear address map can degrade memory utilization and system performance when an access pattern is not linear. To improve memory system performance, the adaptive bank-interleaved linear address map for a DRAM technology is proposed. In our approach, the addresses are efficiently rearranged using the bank-flipping technique for a given application and a memory configuration. The system can configure the address map based on the bank interleaving metric in the systematic way when an application is invoked. Considering image processing applications, the algorithm, the analysis, the design, and the evaluation of the proposed address map are presented. The experimental results show that the presented method can effectively improve the performance with a moderate hardware cost.

Introduction HER2 mutations are found in 2% to 4% of non–small-cell lung cancer cases and are usually mutually exclusive with other genetic alterations. We screened a large cohort of patients from multiple institutions in Korea, described the characteristics of HER2-mutant cases, and reported on several patients who were treated with pan-HER inhibitors. Patients and Methods The study population consisted of 360 patients diagnosed with adenocarcinoma from 4 institutions in Korea from June 2015 to September 2016. Tissue specimens from all participants were screened by direct sequencing, and next-generation sequencing was conducted only on specimens that were positive in direct sequencing. HER2-targeted therapy, either poziotinib or afatinib, was orally administrated. Results Next-generation sequencing was conducted in 129 patients, and finally 29 (8.1%) patients with HER2 mutation were identified. Most patients were female (58.6%), had never smoked (70.0%), and had stage IV non–small-cell lung cancer (55.2%). For all patients, the histologic type was adenocarcinoma, with no coexisting EGFR or ALK alterations. The most common type of HER2 mutation (48.3%) was c.2326_2327insTGT in exon 20. A partial response was observed in 2 patients who received poziotinib and 1 patient who received afatinib. The main toxicities of the pan-HER inhibitors were nausea, diarrhea, and mucositis. Conclusion HER2 mutation was estimated at a frequency of approximately 8.1% in Korean patients with adenocarcinoma in the absence of known driver mutations. Because some of the HER2-mutant cases responded to poziotinib or afatinib, further studies are warranted.

Background EGFR mutation testing is an essential step for the therapeutic decision in newly diagnosed advanced NSCLC patients and usually performed by using DNA extracted from biopsied tumor tissue with a turnaround time(TAT) of 2-3 weeks. Plasma liquid biopsy using ctDNA is minimally invasive and less time-consuming, but shows limited usefulness due to low sensitivity. We conducted a prospective study to demonstrate clinical usefulness of bronchoalveolar lavage fluid (BALF) liquid biopsy using extracellular vesicle(EV)-derived DNA in advanced NSCLC patients by comparing the sensitivity and TAT with conventional tissue biopsy and plasma liquid biopsy Methods The analysis set for liquid EGFR genotyping is 224 BALF samples and 110 plasma samples obtained from newly diagnosed 224 advanced non-squamous NSCLC patients. EGFR genotyping was done through peptide nucleic acid(PNA)-mediated real-time PCR after extracting BALF EV DNA and plasma cfDNA separately. The sensitivity, specificity, and concordance rate of BALF liquid biopsy were calculated in comparison to matched tissue genotyping and plasma liquid biopsy. In addition, TAT was compared between BALF liquid biopsy and tissue genotyping. Results Tissue genotyping revealed 93 EGFR mutant cases (41.5%) and 131 wild-type cases (58.5%). The sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of BALF liquid biopsy to tissue genotyping were 97.8%, 96.9%, 96.8%, 98.4%, and 97.7%, respectively. BALF liquid biopsy showed almost identical performance with standard tissue genotyping, while only 5 cases were mismatched each other. On the other hand, plasma liquid biopsy using cfDNA (n=110) revealed 48.5% sensitivity, 86.3% specificity, and 63.6% concordance rate in comparison to tissue genotyping. The mean turnaround times of BALF liquid biopsy was significantly shortened to 2.6±2.0 days in contrast to 13.9±12.4 days of tissue genotyping. (p Conclusion BALF liquid biopsy can be developed as a novel platform for EGFR mutation testing in advanced NSCLC with almost identical performance with conventional tissue genotyping and significantly shorten TAT to 1-2 days. Citation Format: In Ae Kim, Jae Young Hur, Hee Joung Kim, Wan Seop Kim, Kye Young Lee. BALF liquid biopsy provides a novel platform for speedy and accurate EGFR mutation testing in advanced NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1196.

Despite the development of molecular targeted therapies, few advances have been made in the treatment of lung squamous cell carcinoma (SCC). SOX2 amplification is one of the most common genetic alterations in SCC. Here, we investigated the effects of THZ1, a potent cyclin-dependent kinase 7 (CDK7) inhibitor that plays a key role in gene transcription, in SCC. Lung SCC-derived cell viabilities were assessed using a CCK-8 assay. SOX2 expression and RNAPII-CTD phosphorylation levels after THZ1 treatment were determined by Western blotting. The effect of SOX2 suppression using shRNA was assessed by flow cytometry. Gene expression patterns after THZ1 treatment of lung SCC-derived cells were identified using microarray-based mRNA profiling. We found that THZ1 treatment led to suppression of cell growth and apoptotic cell death in SOX2-amplified SCC-derived cells only, whereas the modest growth-inhibitory effect of cisplatin did not differ according to SOX2 amplification status. We also found that THZ1 decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II and the expression of several genes. Specifically, we found that the expression of transcription-associated genes, including SOX2, was down-regulated by THZ1 in SOX2-amplified SCC cells. This inhibition of SOX2 expression resulted in suppression of the growth of these cells. From our data, we conclude that THZ1 may effectively control the proliferation and survival of SOX2-amplified SCC cells through a decrease in global transcriptional activity, suggesting that CDK7 inhibition leading to transcription suppression may be a promising therapeutic option for lung SCC with a SOX2 amplification.

Background EGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma. Methods Fifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method. Results Among 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides. Conclusions Liquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions. Electronic supplementary material The online version of this article (10.1186/s12885-018-5138-3) contains supplementary material, which is available to authorized users.