Author of

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  P3.09 - Pathology (Not CME Accredited Session) (ID 975)

  • Event: WCLC 2018
  • Type: Poster Viewing in the Exhibit Hall
  • Track:
  • Presentations: 2
  • Moderators:
  • Coordinates: 9/26/2018, 12:00 - 13:30, Exhibit Hall

  • 27586

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    P3.09-10 - Circulating Cell-Free DNA (cfDNA) Molecular Profile of Thai NSCLC Patients Using Difference Variant Frequency of NGS (ID 13744)

    12:00 - 13:30  |  Presenting Author(s): Nareenart Iemwimangsa
    • Abstract
    • Slides

    Background
    

    Detecting cfDNA in the liquid biopsy has become a promising method to explore the genetic landscape of tumor heterogeneity. We developed a pilot-study to find the suitable cutoff of variant frequencies detected from liquid biopsy by NGS to track tumor-associated mutations in NSCLC patients.

    a9ded1e5ce5d75814730bb4caaf49419 Method
    

    Ninety-four samples (24 early-stage NSCLC, 70 late-stage NSCLC) were collected from Ramathibodi Hospital, Thailand. Profiling cfDNA using Ion Proton NGS platform. Overall average base coverage depth from NGS was 10,000x, all variants selected have read depths >10x in order to reach 0.1% sensitivity. Each of selected variants has threshold variant quality (QUAL) >20. Droplet digital PCR (ddPCR) was performed for EGFR-mutation testing to determine the appropriated cutoff variant frequency from NGS.

    4c3880bb027f159e801041b1021e88e8 Result
    

    In early-stage NSCLC, a minimum-threshold variant frequencies at 0.1% could detect EGFR exon19 deletion in all samples (24,100%), with BRAF (12,50%), KRAS (21,87.5%) and other mutations in AKT1, MET, PIK3CA, PTEN, ROS1 (14,58%). None of these mutations identified when using conventional level cutoff at 3% (Table1). ddPCR observed EGFR-mutations in 2 early-stage cases only (8.3%). In late-stage NSCLC, 64 (91.4%) cases were observed multiple mutations, suggesting tumor heterogeneity. At 0.1% cutoff in NGS, Thirty-six (52.9%) cases of EGFR-mutations in NGS and ddPCR were identical. Thirteen (18.6%) samples shown partial discrepancies in the mutations. Interestingly, NGS found EGFR-mutations in 20 (28.6%) samples which ddPCR failed to detect, 12 of them contained T790M. Only one sample (1.4%) using 0.1% cutoff was unable to detect EGFR-mutation. Higher variant allele frequencies were found in EGFR-positive detected by ddPCR compared to not-detected by ddPCR.

    Table1

    Mutations detected		variant allele frequencies detected from liquid biopsy by NGS at minimum threshold cut-off 0.1%	variant allele frequencies detected from liquid biopsy by NGS at minimum threshold cut-off 0.1%	variant allele frequencies detected from liquid biopsy by NGS at conventional level detection of somatic variants cut-off 3%	variant allele frequencies detected from liquid biopsy by NGS at conventional level detection of somatic variants cut-off 3%	variant allele frequencies detected from liquid biopsy by ddPCR (EGFR only)	variant allele frequencies detected from liquid biopsy by ddPCR (EGFR only)
    		N (%)	median (range)	N (%)	median (range)	N (%)	median (range)
    BRAF (V600E)	early stage total N=24	12 (50%)	0.8 (0.3-2.5)	0 (0%)	0	NA	NA
    BRAF (V600E)	late stage total N=70	11 (15.7%)	0.6 (0.1-1.1)	0 (0%)	0	NA	NA
    KRAS	early stage	21 (87.5%)	0.1 (0.1-0.5)	0 (0%)	0	NA	NA
    KRAS	late stage	50 (71.4%)	0.2 (0.1-13.6)	3 (4.3%)	11.1 (5.8-14.3)	NA	NA
    AKT1 (E17K)	early stage	7 (29.2%)	0.1 (0.1-0.7)	0 (0%)	0	NA	NA
    AKT1 (E17K)	late stage	12 (17.1%)	0.1 (0.1-0.7)	0 (0%)	0	NA	NA
    MET exon 14 splicing	early stage	4 (16.7%)	0.1 (0.1-0.1)	0 (0%)	0	NA	NA
    MET exon 14 splicing	late stage	3 (4.3%)	0.2 (0.2-0.2)	0 (0%)	0	NA	NA
    PIK3CA	early stage	9 (37.5%)	0.2 (0.1-0.8)	0 (0%)	0	NA	NA
    PIK3CA	late stage	19 (27.1%)	0.3 (0.1-0.7)	0 (0%)	0	NA	NA
    PTEN (R233*)	early stage	6 (25.0%)	0.1 (0.1-0.4)	0 (0%)	0	NA	NA
    PTEN (R233*)	late stage	11 (15.7%)	0.1 (0.1-0.2)	0 (0%)	0	NA	NA
    ROS1	early stage	0 (0%)	0	0 (0%)	0	NA	NA
    ROS1	late stage	4 (5.7%)	0.55 (0.1-0.7)	0 (0%)	0	NA	NA
    EGFR Exon 19 Deletion	early stage	24 (100%)	0.35 (0.1-2.1)	0 (0%)	0	1 (4.2%)	0.5 (0.5-0.5)
    EGFR Exon 19 Deletion	late stage	25 (35.7%)	0.6 (0.1-49.0)	6 (8.6%)	9.4 (4.5-49.5)	20 (28.6%)	0.65 (0-49.0)
    EGFR L858R	early stage	5 (20.8%)	0.2 (0.1-0.5)	0 (0%)	0	0 (0%)	0
    EGFR L858R	late stage	21 (30%)	1.4 (0.1-9.7)	4 (5.7%)	4.6 (4.4-6.4)	14 (20%)	1.8 (0.3-9.7)
    EGFR T790M	early stage	8 (33.3%)	0.1 (0.1-0.2)	0 (0%)	0	0 (0%)	0
    EGFR T790M	late stage	30 (42.9%)	0.1 (0.1-4.6)	2 (2.9%)	7.5 (5.3-9.7)	16 (22.9%)	0.15 (0-4.1)
    EGFR Exon18 (G719X)	early stage	6 (25%)	0.2 (0.1-0.4)	0 (0%)	0	1 (4.2%)	0.4 (0.4-0.4)
    EGFR Exon18 (G719X)	late stage	4 (5.7%)	4.5 (0.1-49.2)	2 (2.9%)	27.9 (6.7-49.2)	2 (2.9%)	25.6 (2-49.2)
    EGFR Exon 20 Insertion	early stage	0 (0%)	0	0 (0%)	0	0 (0%)	0
    EGFR Exon 20 Insertion	late stage	1 (1.4%)	73.8 (73.8-73.8)	1 (1.4%)	91.7 (91.7-91.7)	0 (0%)	0

    8eea62084ca7e541d918e823422bd82e Conclusion
    

    Detecting variant frequencies at 0.1% could reveal more hidden tumor-associated mutations compared to variant frequency cutoff at 3%. With a careful validation, profiling cfDNA using NGS can be a crucial method to accurately select treatment for NSCLC patients in the future.

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  • 27597

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    P3.09-19 - Matched Thai Lung Cancer Patients Tissue and cfDNA Molecular Profile by NGS (ID 14341)

    12:00 - 13:30  |  Author(s): Nareenart Iemwimangsa
    • Abstract
    • Slides

    Background
    

    Liquid biopsy is the new non-invasive technology to explore the molecular profile. We evaluate molecular alteration from matched tissue and liquid specimen in NSCLC patients using NGS.

    a9ded1e5ce5d75814730bb4caaf49419 Method
    

    A total 61 matched tumors and cfDNA in NSCLC patients were retrieved for DNA extraction. All qualified samples were analyzed by using Next Generation Sequencing (NGS) with Gene read Qiagen Lung Cancer Panel sequencing 45 Genes on Ion Torrent system.Variants from NGS with coverage of higher than 1000X of tissues and 10000X of liquid biopsy, cutoff at 3% variant frequency were considered positive. Each detected mutation was validated by the different method. EGFR-mutation detected at this cutoff was validated by Real-time PCR technique using the ARMS-PCR (Amoy DX Kit) for tissue samples and droplet-digital PCR (ddPCR) for blood samples in all samples.

    4c3880bb027f159e801041b1021e88e8 Result
    

    This study found 59.0% and 19.7% of EGFR mutation, 14.75% and 8.20% of KRAS mutation in tissues and cfDNA, respectively. Moreover, we found 3.29% of BRAF V600E, 1.64% of MET exon14 splice site, and 1.64% of ROS1 mutation in tissue NGS and also confirmed by the other techniques, but there was none of these mutations in the blood sample. Looking at EGFR-mutation detected by different techniques, higher sensitivity, specificity, positive-predictive value, negative-predictive values, and concordant rate were found in tissue NGS and liquid ddPCR compared with liquid NGS at cutoff 3% of variant frequency detection, when the gold standard for validation was ARMS-PCR in tissue testing (Table1).

    Table1. Performance of NGS, ARMS and ddPCR for EGFR mutation detection in matched tissue and cfDNA in lung cancer patients.

    Tissue by ARMS PCR Result Tissues by NGS negative positive total Sensitivity= 87.7% negative 14 11 25 Specificity= 100% positive 0 36 36 Positive predictive value= 100% Total 14 47 61 Negative predictive value= 75.6% Concordance = 82% Tissue by ARMS PCR cfDNA by NGS negative positive total Sensitivity= 35.7% negative 12 37 49 Specificity= 98.2% positive 2 10 12 Positive predictive value= 97.9% Total 14 47 61 Negative predictive value= 38.9% Concordance = 42.6% Tissue by ARMS PCR cfDNA by ddPCR negative positive total Sensitivity= 82.7% negative 14 14 28 Specificity= 100% positive 0 33 33 Positive predictive value= 100% Total 14 47 61 Negative predictive value= 69.4% Concordance = 77.05% Tissue by NGS cfDNA by NGS negative positive total Sensitivity= 36% negative 20 29 49 Specificity= 93.2% positive 5 7 12 Positive predictive value= 84.8% Total 25 36 61 Negative predictive value= 55.8% Concordance = 60.66% cfDNA by ddPCR cfDNA by NGS negative positive total Sensitivity= 45.5% negative 25 24 49 Specificity= 97.7% positive 3 9 12 Positive predictive value= 94.5% Total 28 33 61 Negative predictive value= 65.6% Concordance = 65.58%

    8eea62084ca7e541d918e823422bd82e Conclusion
    

    Using tissue for molecular profile testing is still being the gold standard testing. Liquid biopsy is less invasive, but in our study, liquid NGS performed less sensitivity, specificity, PPV, NPV, and concordance rate compared with tissue NGS. We need to explore more for proper cutoff of variant allele frequency to develop more sensitivity and specificity of liquid NGS.

    6f8b794f3246b0c1e1780bb4d4d5dc53

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