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In Ae Kim



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    MA25 - Precision Medicine in Advanced NSCLC (ID 352)

    • Event: WCLC 2019
    • Type: Mini Oral Session
    • Track: Advanced NSCLC
    • Presentations: 1
    • Now Available
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      MA25.07 - Genomic Profiling of Lung Adenocarcinoma by Targeted NGS Using Extracellular Vesicle-Derived DNA in Bronchoalveolar Lavage Fluid (Now Available) (ID 1532)

      14:30 - 16:00  |  Author(s): In Ae Kim

      • Abstract
      • Presentation
      • Slides

      Background

      Extracellular vesicles (EV) are membrane-bound and nanometer-sized particles shed from most types of cells in our body and found in circulation, containing double-stranded genomic DNA reflecting mutational status of the parental tumor cells. Recently, we demonstrated that EVs successfully isolated from bronchoalveolar lavage fluid (BALF) in non-small cell lung cancer (NSCLC) patients. Several studies also have shown that EV-derived DNA is superior to cfDNA in plasma for detection of mutations in NSCLC and pancreatic cancer. We identified that liquid biopsy for EGFR genotyping using EV-derived DNA in BALF showed almost 100% sensitivity with tissue typing in advanced NSCLC patients. Therefore, we hypothesized that targeted next–generation sequencing (NGS) using EV-derived DNA in BALF may correlate with results using tissue in patients with EGFR-mutated lung adenocarcinoma.

      Method

      To address this hypothesis, we compared with the targeted NGS profile using between BALF EV-derived DNA and tissue DNA in 20 patients with EGFR-mutated lung adenocarcinoma. Four types of somatic variants (SNVs, small indels, CNVs and gene fusions) of BALF-EV or FFPE tissue samples were analyzed by CancerSCANTM, a capture-based targeted sequencing platform, which targets 375 genes covering about 2.5-megabase genomic regions including full CDSs of 374 genes, selected intronic regions of 23 genes for fusion detection, and 1kb TERT promoter region.

      Result

      Targeted sequencing resulted in over 99% of the target regions covered at a mean depth of 190-750× except one sample. DNA yields were higher in tissue DNA than EV-derived DNA (827.02ng vs 89.10ng). Depth of coverage (753x vs 379x) and estimated tumor purity (53% vs 23%) were also higher in tissue DNA than EV-derived DNA. However, estimated library size was not significantly different between tissue DNA and EV-derived DNA (50G vs 47G) and fragment size of DNA were longer in EV-derived DNA than tissue DNA (175.5bp vs 169.5bp). These findings support that EV-derived DNA has sufficient quality and quantity for NGS. By using mutations detected in tissue DNA as a reference, we achieved 83% sensitivity for somatic and clinically significant variants in EV-derived DNA. Clinically significant mutations in EGFR, TP53, PTEN, APC, JAK3 and PIK3CA were identified with an overall concordance of 81% in matched tissue DNA and EV-derived DNA. Variants in EGFR and TP53 were most common, with concordance of 80% and 100%, respectively. Variant allele frequencies of EGFR and TP53 were most abundant in range of 10-25% in tissue DNA, while much lower (<5%) in EV-derived DNA. Tumor mutation burdens (TMB) of EV-derived DNA showed correlation with tissue DNA (R2=0.21).

      Conclusion

      To our knowledge, this is the first of study of comprehensive clinical NGS panel using EV-derived DNA of BALF and matched tumor tissue biopsies in patients with lung adenocarcinoma. Although EV-derived DNA demonstrated comparable results to tissue DNA, it is needed much higher sequencing coverage and optimization of NGS-pipeline to detect low-allele frequency variants of EV-derived DNA. This study demonstrates the feasibility and clinical utility of BALF EV-derived DNA for patients with lung adenocarcinoma.

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    P1.01 - Advanced NSCLC (ID 158)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Advanced NSCLC
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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      P1.01-32 - Bronchoalveolar Lavage as an Alternative to Rebiopsy for Detection of T790M Mutation in NSCLC Patients with Acquired Resistance to EGFR-TKIs (ID 2164)

      09:45 - 18:00  |  Author(s): In Ae Kim

      • Abstract

      Background

      Rebiopsy is current standard to detect T790M mutation, but has limited value due to inaccessibility in significant number of patients. Plasma liquid biopsy using ctDNA has been adopted, but the sensitivity is not satisfactory. Extracellular vesicles (EVs) have been proven to contain double-stranded genomic DNA reflecting mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC). Accordingly, EVs can be translated into clinically useful liquid biopsy for EGFR genotyping using bronchoalveolar lavage fluid (BALF) obtained from the tumor site. We investigated the role of BALF for detecting T790M mutation in the patients who developed acquired resistance to EGFR-TKI by comparing with standard tissue rebiopsy and liquid biopsy using plasma ctDNA.

      Method

      Forthy-eight EGFR mutation-positive NSCLC patients with acquired EGFR-TKI resistance were evaluated respectively. EVs were isolated from BALF by ultracentrifugation and EVs DNA extracted after eliminating free-floating DNA. PNA clamping-assisted fluorescence melting curve analysis (PANAMutyper™) were used to assess the EGFR mutation status in rebiopsy tissue, plasma and BALF.

      Result

      Median age of 48 patients was 63.5 years (range, 44-87 years); 26 (52.1%) were women; and 27 (56.3%) were never-smokers. Thirty-three of 48 patients (62.5%) underwent a repeated biopsy with results as follows: there was insufficient tissue in 3 of 33 (9.1%) and T790M mutation in 12 of 30 patients who were pathologically confired as NSCLC (40.0%). Fifteen of 48 patients (31.3%) did not undergo a repeated biopsy at progression for the following reasons: patient with target lesion less than 10 mm in 6 cases; and no lesion amenable to biopsy such as leptomeningeal seeding, distant metastasis to bone or adrenal gland and lung nodules which is difficult to agmqccess in 9 case. The EGFR mutation-positive rate from BALF (37.5%) was higher than that determined from plasma or tissue at 16.7% and 25.0%, respectively. Interestingly, out of the 36 patients who were not able to undergo a repeated biopsy, whose biopsy results were insufficient for diagnosis or who did not have T790M mutation, five (10.4%) and three (6.3%) samples were additionally positive from the BALF and plasma test, respectively.

      Conclusion

      Less invasive genotyping by PNA clamping-assisted fluorescence melting curve analysis with EVs DNA extracted from BALF is a promising approach to the detection of T790M mutation during EGFR TKI treatment.

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    P2.17 - Treatment of Early Stage/Localized Disease (ID 189)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Treatment of Early Stage/Localized Disease
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.17-25 - Genomic Insight into Stage I Pulmonary Adenocarcinoma for Recurrence by Targeted Next-Generation Sequencing Analysis (ID 2317)

      10:15 - 18:15  |  Presenting Author(s): In Ae Kim

      • Abstract

      Background

      Despite surgical resection, stage I pulmonary adenocarcinoma has a recurrence rate of 18.4%–25%. Prognostic markers of localized lung adenocarcinoma remain undefined. Targeted next-generation sequencing (NGS) is supposed to be useful to identify recurrence-related genes. We aimed to identify genes and co-occurring mutations that might predict recurrence after surgical resection of stage I pulmonary adenocarcinoma through targeted NGS.

      Method

      Tissues from 202 patients who had complete resection of stage I pulmonary adenocarcinoma at Konkuk University Medical Center, from 2005 to 2017 (median follow-up: 48 months), were analyzed by targeted NGS using a panel of the 170 cancer-related genes (KF1 panel by Macrogen Inc. Korea,). We compared RFS (relapse-free survival) according to frequent genetic alterations using the Kaplan-Meier method and compared their combinations to identify recurrence-related genes by multivariate analysis.

      Result

      The most frequent genetic alterations were EGFR (56.9%), TP53 (37.7%), KRAS (13.4%), and PIK3CA (10.4%) in surgically resected stage I pulmonary adenocarcinoma. In the tumors with single gene mutation, EGFR mutation was a good prognostic factor and TP53 mutation was a poor prognostic factor for recurrence. (EGFR mutation, HR 0.26, 95% CI 0.07-0.95, p=0.024 vs TP53 mutation, HR 3.39, 95% CI 1.17-9.8, p=0.02). In the tumors with two gene mutations, similar tendency has been observed but there was no statistical significance. Mutation of CTNNB1 was significantly related to recurrence in multivariate analysis (HR3.76, P=0.003) and this finding suggests the CTNNB1 as a molecular biomarker for recurrence by NGS method. TP53 and KRAS combination mutations tended to be associated with shorter RFS (HR=1.89, p=0.08). Recurrence was not associated with the number of the co-occurring mutations but with their specific combinations such as the combination of CTNNB1 and EGFR.

      Conclusion

      The CTNNB1 mutation is associated with shorter RFS as compared with wild-type CTNNB1 in resected stage I adenocarcinoma. Larger datasets are required to validate whether the CTNNB1 gene is an independent predictive factor of early stage adenocarcinoma recurrence. NGS can be useful tool to predict the risk of recurrence and select the patient to need the adjuvant chemotherapy in stage I adenocarcinoma.