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Rayleen Bowman
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MA23 - Preclinical Models and Genetics of Malignant Pleural Mesothelioma (ID 353)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Mesothelioma
- Presentations: 1
- Now Available
- Moderators:Ramon Palmero Sánchez, Raphael Bueno
- Coordinates: 9/10/2019, 14:30 - 16:00, Copenhagen (1980)
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MA23.09 - Fusion Genes Identified from Whole Genome and Whole Transcriptome Sequencing of Malignant Pleural Mesothelioma Tumours (Now Available) (ID 2014)
14:30 - 16:00 | Author(s): Rayleen Bowman
- Abstract
- Presentation
Background
Malignant Pleural Mesothelioma (MPM) is an asbestos-related cancer without curative treatment. Fusion genes result from structural chromosomal rearrangements such as translocation, inversion, amplification and deletions, leading to erroneous apposition of components of two or more genes. Consequences include abolition of gene functions that protect against tumourigenesis, or increased activation of genes that promote cell proliferation. To identify fusion genes in MPM genomes, we executed whole genome sequencing (WGS) on eight MPM tumours, and validated the expression of putative fusion genes identified from WGS by whole transcriptome analysis (RNA-Seq).
Method
Histology of eight MPM tumours was confirmed by two qualified anatomical pathologists, prior to extraction of genomic DNA and RNA. Whole genome and whole transcriptome sequencing were performed using Illumina HiSeq platforms. Following stringent data processing and filtration, putative fusion variants were called using an in-house bioinformatics pipeline. Fusion events with potential functional consequences were then validated by whole transcriptome analysis, and annotated using TCGA Fusion Gene Data Portal and The Gene Ontology Resource.
Result
A total of 592 and 321 putative fusion variants were called respectively from WGS data using Delly, and from RNA-Seq using STAR-Fusion computational tools. Expression of WGS putative fusion variants was confirmed in RNA-Seq data, resulting in twelve fusion genes being identified. Among 24 genes involved in fusion events, twenty-two were listed in TCGA Fusion Gene Data Portal with gene partners that were not identified in our cases. Two genes were novel to that database. Multiple functional processes that may lead to tumour development were attributable to these genes including protein polyubiquitination, protein deubiquitination, antioxidant activity, DNA repair, immune response, integrin-mediated signalling pathway, chromatin organization, transcription coactivator activity, angiogenesis, natural killer cell proliferation and DNA-binding transcription factor activity.
Conclusion
In combination, WGS and RNA-Seq data analysis revealed several fusion genes that warrant further investigation as possible drivers of malignant mesothelioma, and which may serve as diagnostic and therapeutic targets.
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P1.03 - Biology (ID 161)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.03-11 - Molecular Testing of Small Bronchoscopy Specimens Using NanoString Technology (ID 274)
09:45 - 18:00 | Author(s): Rayleen Bowman
- Abstract
Background
Molecular testing for driver variants in oncogenes is crucial for NSCLC management to predict response to targeted therapy. In the majority of cases, NSCLC is diagnosed by trans-thoracic needle aspiration or more commonly bronchoscopy techniques resulting in small diagnostic tissue biopsies or cytological samples. As such specimens may be inadequate for molecular testing, we tested the accuracy of a novel digital molecular barcoding assay to detect actionable mutations in a single-centre cohort.
Method
A consecutive cohort of 46 specimens (19 endobronchial biopsy, EnBx; 18 transbronchial biopsy, TBBx; seven bronchoalveolar lavage, BAL; and two transbronchial needle aspirate, TBNA) were obtained ancillary to primary diagnostic specimens from 36 patients undergoing EBUS-guided bronchoscopy at The Prince Charles Hospital. Specimens containing at least 5ng DNA after standard column-based extraction methods were analysed using the NanoString SNV Solid Tumour Panel for testing of 104 somatic variants across 25 genes of clinical significance. NanoString variants calls were compared with routine clinical testing results from the primary diagnostic sample. Agreement analyses for variants common to both methods revealed the positive, negative and overall percentage agreement (PPA, NPA, OPA). One discordant case was validated using droplet digital PCR.
Result
Using NanoString, molecular analysis was feasible for 60.1% (28/46) of specimens. At least one variant was identified in 8/28 (28.6%) cases (Table 1). Two (7.1%) cases harboured dual mutations. KRAS mutations were detected in six (21.4%) cases, and EGFR in two (7.1%). Two patients would be eligible for targeted therapy. Agreement analysis for the two methods revealed PPA, NPA and OPA of 100%, 88.9% and 92.3%. In one discordant case, NanoString identified a KRAS G12C mutation and was confirmed by ddPCR with a mutant allele frequency of 5.5%. The mean time for reporting clinical mutation test results was 19.6 days. Of the 18 excluded cases with insufficient DNA, five had routine testing results for comparison however 3/5 cases cited insufficient DNA for reliable EGFR testing.
Table 1: Clinical and molecular characteristics of bronchoscopy samples used for molecular testing Clinical molecular testing
NanoString
Concordance
Case no.
Sample type
Histological classification
DNA yield (μg)
Mutation testing result (MAF)
Mutation testing method
TAT to result (days)
SNV panel result
Agreement (Yes/No)
1
BAL
No evidence of malignancy
2.81
Not performed
N/A
N/A
WT
NCA
2
TBNA
AC
4.39
Not performed
N/A
N/A
WT
NCA
3
TBBx
SCC
0.39
Not performed
N/A
N/A
KRAS G12R
NCA
4
TBBx
AC
1.82
EGFR exon 19 (L747_P753>S) del (24%)
NGS TruSight
21
EGFR exon 19 (L747_P753>S) del
Yes
5
TBBx
No evidence of malignancy
1.24
Not performed
N/A
N/A
WT
NCA
6
TBBx
No evidence of malignancy
0.62
Not performed
N/A
N/A
WT
NCA
7
EnBx
AC
1.41
WT for EGFR, KRAS, NRAS, BRAF
NGS TruSight
13
WT
Yes
8
EnBx
SCC
0.57
Not performed
N/A
N/A
WT
NCA
9
TBBx
SCC
0.53
Not performed
N/A
N/A
WT
NCA
10
TBBx
AC
0.51
WT for EGFR, KRAS, NRAS, BRAF
NGS TruSight
20
KRAS G12C
No*
11
TBBx
AC
0.31
EGFR L858R (12%), EGFR T790M (6%)
NGS TruSight
13
EGFR L858R, EGFR T790M
Yes
12
TBBx
No evidence of malignancy
0.69
Not performed
N/A
N/A
WT
NCA
13
TBBx
NSCLC
4.26
WT for EGFR
castPCR
48
WT
Yes
14
EnBx
AC
1.37
KRAS G12C (10%)
NGS TruSight
23
KRAS G12C
Yes
15
EnBx
NSCLC
19.6
KRAS G12A (25%)
NGS TruSight
20
KRAS G12A
Yes
16
EnBx
Carcinoid
1.16
Not performed
N/A
N/A
WT
NCA
17
TBBx
SCC
5.4
Not performed
N/A
N/A
WT
NCA
18
EnBx
AC
4.03
WT for EGFR, KRAS, NRAS, BRAF
NGS TruSight
19
WT
Yes
19
EnBx
SCC
2.12
Not performed
N/A
N/A
WT
NCA
20
TBBx
AC
3.85
WT for EGFR
castPCR
14
KRAS G12C, NRAS Q61K
NCA
21
EnBx
SCC
6.4
Not performed
N/A
N/A
WT
NCA
22
TBBx
AC
1.63
WT for EGFR
castPCR
20
KRAS G12C
NCA
23
EnBx
AC
0.234
WT for EGFR, KRAS, NRAS, BRAF
NGS TruSight
16
WT
Yes
24
BAL
AC
0.871
BRAF G466V (12%)
NGS TruSight
24
WT
Yes
25
TBBx
AC
0.0918
BRAF G466V (12%)
NGS TruSight
N/A
WT
Yes
26
TBBx
AC
0.893
WT for EGFR
castPCR
13
WT
Yes
27
TBBx
SCC
0.66
Not performed
N/A
N/A
WT
NCA
28
EnBx
AC
4.4
WT for EGFR
castPCR
10
WT
Yes
Mean
2.58
19.6
The performance of the NanoString platform for SNV characterisation was highly concordant with alternate clinical testing methods for those with sufficient DNA. Advantages of NanoString include its multiplex capacity, high sensitivity, low nucleic acid input, reduced turn-around time (<24hr) compared to alternate testing methods. The NanoString platform is a robust method for identification of actionable variants in NSCLC where at least 5ng of DNA is available.