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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
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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
      • Slides

      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

      Conclusion

      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.