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Cristina Teixidó
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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 - 16:00 | Author(s): Cristina Teixidó
- 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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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-014 - Simultaneous Gene Profiling of Advanced NSCLC: Single-Molecule Quantification of DNA and RNA by nCounter3D™ Technology (ID 9808)
09:30 - 16:00 | Presenting Author(s): Cristina Teixidó
- Abstract
Background:
Currently, assessment of several tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). Tools for molecular profiling are based on DNA, RNA and protein (PCR, NGS, FISH, IHC). However, these tests have several disadvantages including hands-on-time and tissue mass requirements. Nanostring digital barcoding technology enables simultaneous assay of different analytes, DNA and RNA from a single sample with 3D Biology Technology.
Method:
The nCounter Vantage 3D SNV:Fusions Lung Assay was used to analyze a total of 36 formalin-fixed paraffin embedded (FFPE) samples from advanced NSCLC patients. Samples were known to harbor mutations (EGFR, KRAS, NRAS, PIK3CA, BRAF, P53) or gene-fusion rearrangements (ALK, ROS1, RET, NTRK1) as verified by sequencing (Ion Torrent, Gene Reader), nCounter Elements, IHC and/or FISH. Probes were designed to target 25 genes for SNVs (104 different point and InDel mutations) as well as four genes for fusion transcripts (ALK, ROS1, RET, NTRK1) including 33 specific variants. The 3D workflow requires pre-amplification of gDNA, whereas RNA does not require any enzymatic treatment. After hybridization, the analytes (DNA/RNA) are pooled for simultaneous, single-lane, digital counting in total turnaround of 3 days. A total amount of 5ng DNA and 150ng RNA from two4-micron FFPE-sections was used for the assay without microdissection.
Result:
A total of 72 analyses (DNA/RNA) were performed with an evaluation pass of 97.2% (70/72 analyses yielded results) and 89% concordant results (64/72). Sensitivity of the technique was 92.1%. Among 41 SNVs interrogated in this study 34 were successfully detected (two not evaluable). Five new mutations were found involving NRAS, FBXW7, GNA11, FGFR2 and KRAS genes. Of those, only two were considered false positives as they were not confirmed by alternative sequencing and/or PCR. The remaining three were not assessable for test confirmation. For gene fusion analysis, 13 known positive samples were tested. All fusion transcripts were detected for ALK (n=5) RET (n=2) and NTRK1 (n=1). For ROS1 (n=5) there were 2 false negatives only detected by nCounter Elements target-specific assay.
Conclusion:
We have shown that the SNV detection chemistry can be successfully combined with fusion gene expression analysis by using the nCounter 3D™ single-workflow. The Nanostring nCounter Vantage 3D SNV:Fusions Lung Assay is highly efficient in detecting hotspot mutations as well as gene rearrangements. The assay is simple, features a brief hands-on time and requires low amounts of genomic material, supporting minimal use of precious samples.
