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P. Jares
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P1.01 - Advanced NSCLC (ID 757)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.01-075 - Simultaneous Multiplex Profiling of Gene Fusions, METe14 Mutations and Immune Genes in Advanced NSCLC by NCounter Technology (ID 9481)
09:30 - 09:30 | Author(s): P. Jares
- Abstract
Background:
Assessment of several immune-genes and tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). We have previously demonstrated the ability of the transcript-based nCounter Technology for the detection of ALK, ROS1 and RET gene fusions, using a customized codeset (Reguart et al. Clinical Chemistry 2017). Here, we present the first results of the validation in advanced NSCLC samples of a new CodeSet designed to simultaneously characterize clinically relevant gene fusions, MET alterations and the expression of immune genes.
Method:
We have designed an in-house custom set to detect driver fusions involving 4 genes (ALK, ROS, RET, NTRK), MET exon 14 skipping mutation, MET overexpression and immune genes (PD1, PDL-1, CD4, CD8, FOXP3, GZMM, IFNG). A panel of ALK-ROS-RET-NTRK positive cell lines (H2228, H3122, SU-DHL-1, HCC78, BaF3 pBABE, LC2/ad and a NTRK-positive cell line), Hs746T (METex14), EBC-1 (overexpressing MET) and a negative cell line (PC9) were used for the initial validation of the panel. Subsequently, 58 FFPE samples from advanced NSCLC patients, previously characterized by FISH, RT-PCR and IHC, have been analyzed. Total amount of 100-150 ng RNA was used for detection. Workflow includes RNA isolation, hybridization and digital counting with for a total turnaround of 3 days. Raw counts were normalized using positive controls, negative controls and house-keeping genes.
Result:
.Results obtained with the cell lines positive for ALK, ROS1, RET and NTRK1 fusion genes were exactly coincident with their genotypes, with fusion transcripts successfully detected in all cases by 3’/5’ imbalance and direct fusion probes. In addition, METex14 splicing transcripts were detected in the Hs746T cells at significant levels, higher than those of wt MET mRNA. In contrast, METex14 mRNA counts were almost undetectable in the rest of cell lines. Regarding FFPE samples from advanced patients, 46 could be successfully analyzed by nCounter. Among 13 patients positive for ALK and ROS1 fusions, 12 were confirmed by nCounter. Regarding the METex14 splicing variant, 5 out of 6 patients previously detected by RT-PCR were also positive by nCounter.
Conclusion:
Preliminary data suggest that multiplex detection of clinically relevant drivers can be successfully achieved using nCounter Technology. The assay is simple, requires short hands-on-time, needs low input RNA and is highly efficient in detecting gene rearrangements and METex14 splicing variants. Results will be prospectively validated in a larger cohort of advanced NSCLC patients and we will determine if clusters of different inmune-phenotypes exist among oncogenic-driven NSCLC tumors.
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P3.01 - Advanced NSCLC (ID 621)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.01-045 - Correlation of EGFR Mutation Detection in CtDNA by Two Different Platforms in Advanced NSCLC Patients from a Single Institution (ID 9475)
09:30 - 09:30 | Author(s): P. Jares
- Abstract
Background:
Circulating-free tumor DNA (ctDNA) has emerged as a sensitive and feasible non-invasive blood-based approach alternative to tissue biopsies to screen for genetic drivers in advanced non-small cell lung cancer (NSCLC) patients. Recently, the COBAS Mutation Test has been FDA-approved for the detection of EGFR mutation (EGFRm) in peripheral blood but other approaches are currently used in clinical practice. Here, we aimed to correlate two different platforms for EGFRm monitoring ctDNA in an enriched cohort of tissue-genetically phenotyped EGFRm advanced NSCLC patients.
Method:
Blood samples were prospectively obtained from an enriched cohort of EGFR+ advanced NSCLC patients. Formalin-fixed paraffin-embedded (FFPE) tumor was characterized by NGS (Ion AmpliSeq Lung Cancer Research Panel v.2) in all patients at diagnose. Plasma ctDNA derived from peripheral whole blood was evaluated by COBAS EGFR Mutation Test v2 and a Peptide Nucleic Acid (PNA) probe–based real-time polymerase chain reaction blinded to baseline tumor genotype. Diagnostic accuracy and concordance of both blood techniques was used for direct comparison with respect to the molecular status of FFPE tissue.
Result:
A total of 80 matched pairs of peripheral blood samples from 40 patients were collected. Baseline tissue NGS reported mutations at exon 19 del (n=23), exon 21 (n=10), exon 18 (n=2), exon 20 (n=2) and T790M (n=3). Four wild-type EGFR tumors were used as controls. Blood samples were obtained at diagnose (n=12) and during tyrosine-kinase inhibitor (TKi) treatment for monitoring (n=28). Overall, concordance between both blood-based techniques with respect tissue-NGS was 100% (4/4) for negative controls and 55% (20/36) for positive tissue-NGS samples. Detection based on PNA and COBAS was negative in 60% (24/40) and 32.5% (13/40) patients respectively. Among 19 samples negative by PNA at monitoring, COBAS allowed plasma EGFRm detection in 11 patients. At baseline, the only two negative samples patients by both techniques were found in patients with localized brain disease. Six patients had detectable driver T790M mutation; among three patients with T790M+ in tissue, COBAS allowed detection in plasma in one patient whereas none was identified with PNA. The other three patients had acquired T790M mutations identified only in blood, all by COBAS.
Conclusion:
In this prospective blinded validation cohort, both methods retained high specificity. However, major differences between techniques were observed for longitudinal monitoring of EGFRm in blood.
