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  • CT99021 br Targeted DNA and RNA Seq Cohort Characterization

    2020-08-12


    Targeted DNA- and RNA-Seq Cohort Characterization
    The cohort was next characterized using the integrated NSCLC NGS assay (Figure 3; Supplemental Tables S3 and S4). Consistent with other studies such as TCGA, fusions in ALK, RET and ROS1 were restricted to the LUAD subtype. Eight fusions were identified across all specimens: EML4-ALK (N = 2), KIF5B-RET, EZR-ROS1, CD74-ROS1, KIF5B-RET, CCDC6-RET, and CD74-NRG1.
    Figure 3. Multiomic characterization of NSCLC FFPE cohort reveals a spectra of mutations consistent with underlying histological subtype.
    Translational Oncology Vol. 12, No. 6, 2019 An Integrated Next-Generation Sequencing System Haynes et al. 841
    Figure 4. Select mRNA expression markers revealed by targeted RNA-Seq distinguish LUSCs from LUADs. (A) PCA analysis of differentially expressed mRNAs between LUADs (blue) and LUSCs (orange). Comparison of expression distributions of log2 transformed normalized expression (x-axis) for mRNAs differentially expressed between LUADs (blue) and LUSCs (orange) shows similar profiles when comparing (B) our data (targeted RNA-Seq) to C) TCGA (microarray).
    Imbalance of 3′ expression relative to 5′ expression was detected for both ALK fusions and for two of three RET fusions. The one RET fusion for which an imbalance was not detected was in a specimen that fell below the minimum RNA functional copy input of 200 copies. Neither of the ROS1 fusions showed evidence of 3′/5′ imbalance. Three imbalances were detected in samples that were not accompanied by a positive fusion call. Two of these samples, ADC48 (ALK) and SQCC17 (ALK), were otherwise mutation negative. However, the third sample (AD62), which had a RET imbalance, was also positive for EGFR and TP53 mutations. In addition, MET exon 14 skipping was detected in two LUADs, one of which had ~30% skipped and the other with nearly 100% skipped. All gene fusions and MET exon 14 skipping calls were confirmed by an independent assay (see Methods). DNA variant calls were consistent with the underlying histopa-thology and mutation prevalence according to TCGA and COSMIC. For example, mutations in KRAS, EGFR, and STK11 were present in LUADs whereas PIK3CA and FGFR3 were detected in LUSC. TP53 mutations were detected in both subtypes. Also consistent with other NSCLC cohorts, KRAS and EGFR mutations in LUAD specimens were mutually exclusive events [32,38]. When examining the spectrum of KRAS mutations in the LUAD specimens, CT99021 12 was the most frequently mutated (74% of all 62 KRAS mutations), a result that is consonant with other studies such as the TCGA LUAD cohort where codon 12 represented 96% of KRAS mutations [32]. Mutations in other codons of KRAS were largely represented by codons 13 (18%) and 61 (5%). Two unexpected KRAS mutations were detected in LUSC. One specimen, SQ28 (KRAS p.G13D) was identified as a poorly differentiated NSCLC with LUAD features (TTF1+, p40−) and the other, SQCC5, presented with a noncanonical KRAS variant p.N26I at 20% variant allele frequency.
    Integrative analysis of DNA and RNA markers found 10 cases that were positive for targeted RNA-based variants (MET exon 14 skipping or gene fusions). Nine of those 10 cases were negative for any DNA mutation, suggesting that these oncogenic driver events are mutually exclusive. Interestingly, one case was positive for both 
    EML4-ALK and KRAS p.G12D. While ALK fusions are generally thought to be mutually exclusive with other oncogenic mutations such as KRAS and EGFR, these have been previously reported to co-occur in rare instances [39,40].
    Comparative Expression Analysis of Adenocarcinomas and Squamous Cell Carcinomas
    We compared the expression profiles between LUAD and LUSC subtypes to identify mRNAs within those targeted by the panel that were differentially expressed between the two histological subtypes. MSLN, TYMS, CDKN2A, and FGFR2 showed significant differences in expression between subtypes (corrected P b 0.05 and more than two2-fold differences for all four genes, Figure 4), with elevated levels of MSLN in LUADs and elevated levels of CDKN2A, FGFR2, and TYMS in LUSCs. MSLN has been reported to be expressed at higher levels in LUADs relative to LUSCs [41]. Similarly, TCGA data indicate higher levels of CDKN2A, FGFR2, and TYMS in LUSCs [31,32]. Indeed, when comparing our results to TCGA LUAD and LUSC cohorts, we observed qualitatively similar gene expression distributions (Figure 4, B and C). While CDKN2A is on average expressed at higher levels in LUSCs relative to LUADs, the gene is also reported as recurrently downregulated in LUSCs via epigenetic silencing and whole gene deletions [31]. This is consistent with the apparent bimodality of the CDKN2A expression distribution within LUSCs, evident in our data (Figure 4B) and the TCGA LUSC cohorts (Figure 4C). The segregation of LUSCs, largely driven by CDKN2A expression, is also apparent by PCA analysis (Figure 4A). Further co-expression analysis revealed a strong positive correlative relationship between PD-L1 and PD-L2 mRNA expression in both LUAD (SCC: 0.76; P b 2e-16, Figure S1) and LUSC (SCC: 0.66; P