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Somthawin Lukrak
Author of
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P2.01 - Advanced NSCLC (Not CME Accredited Session) (ID 950)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.01-132 - Comparison Molecular Profiles in NSCLC by Using Different NGS Platforms and<br /> Different Variant Frequency Cutoff (ID 14349)
16:45 - 18:00 | Author(s): Somthawin Lukrak
- Abstract
Background
There are several methods to detect molecular alterations in precision medicine era. This is a pilot study aimed to explore the concordance of molecular alterations testing using NGS different platforms and different cutoff allele frequency in NSCLC patients.
a9ded1e5ce5d75814730bb4caaf49419 Method
Ten NSCLC patients’ FFPE were retrieved for DNA extraction. All qualified samples were analyzed using NGS on 45 cancer genes panel on Ion Torrent system NGS Gene read Qiagen Lung Cancer Panel. Variants from NGS with coverage of are higher than 1000X. The cutoff 1% and 3% of variant allele frequency were considered positive. EGFR, BRAF, KRAS were validated by Real-time PCR technique using the Amoy DX and Actionable Insights Tumor Panel in GeneReader NGS system.
4c3880bb027f159e801041b1021e88e8 Result
We found 90%/30% EGFR-mutation, 40% BRAF V600E, 70%/30% KRAS-mutation by NGS using allele frequency cutoff at 1% and 3%, respectively. We validated by Real-Time PCR and Actionable GeneReader showed 70%/40% EGFR-mutation, 20%/10% BRAF V600E, and 0%/30% of KRAS-mutation (Table1). Regarding EGFR-mutation, 5 cases of discordance showed positive at 1% but negative at 3% cutoff allele frequency by NGS and validated by Real-Time showed positive all 5 cases. Two negative cases by Real-Time PCR show positive in NGS cutoff 1% (2/2) and 3% (1/2). In early stage (80%), there was 60% of EGFR 19 del detected by NGS cutoff 1%. The patients have continued follow-up at the clinic with the mDFS of 4.7 years. Two stage IV patients with exon19del were death and received EGFR-TKI as a second-line treatment with mOS at 1.1 and 1.2 years.
Table 1
8eea62084ca7e541d918e823422bd82e ConclusionNumber of 10 cases positive
NGS IonTorrent Cutt Off at 1%
NGS Ion Torrent Cutt Off at 3%
NGS GeneReader
Real-time PCR
EGFR Mutation
9
3
4
7
Exon 19 Del
6
2
2
5
L858R
1
0
2
2
>1 EGFR Mutations
2
1
0
0
No Mutation
1
7
6
3
Different platform of NGS and different cutoff variant allele frequency gave the different result of gene frequency. We need to explore the standard of NGS testing including the proper cutoff for allele frequency in order to establish the most efficient method and correlate with the clinical treatment outcomes.
6f8b794f3246b0c1e1780bb4d4d5dc53
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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: 1
- Moderators:
- Coordinates: 9/26/2018, 12:00 - 13:30, Exhibit Hall
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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 | Author(s): Somthawin Lukrak
- Abstract
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.
6f8b794f3246b0c1e1780bb4d4d5dc53
