br Materials and Methods br Tumor
Materials and Methods
All specimens were obtained from MD Anderson Cancer Center (Houston, TX) under Institutional Review Board–approved study protocols. Core needle biopsies (CNBs) were obtained as a set of FFPE tissue slides in 5-μm or 10-μm sections with 10-30 μm available for each tumor (N = 109). Surgically resected specimens were obtained as 2 × 10–μm sections for each tumor (N = 110). CNBs and surgical resections are summarized in Table 1. Fine-needle aspirate (FNA) smears for 50 BATTLE-2 trial subjects were obtained as sets of one or three slides.
Target Selection and Panel Design
The hybrid DNA/RNA panel consists of an RNA library pool which covers 107 gene fusions recurrent in NSCLC including ALK, RET, and ROS1. DNA hotspots were prioritized according to the mutation prevalence and level of evidence supporting targetable therapy options in NSCLC. The DNA NGS library covers 55 hotspot regions in 20 genes including EGFR, KRAS, STK11, PIK3CA, TP53, and others (Table 2) and is based on previously
Table 1. Clinicopathological Summary of NSCLC Specimens Profiled by Targeted NGS
Core Needle Biopsies Surgical Resections
Histopathology Squamous cell
838 An Integrated Next-Generation Sequencing System Haynes et al. Translational Oncology Vol. 12, No. 6, 2019
Table 2. Content of the Targeted NGS DNA-Seq and RNA-Seq Panel
RNA Pool Content
DNA Pool Content
3’ Fusion Partner # Unique Breakpoints 3′/5’ Additional RNA Targets DNA Targets
53 ● ABCB1 MSLN ALK FGFR3 ROS1
22 ● BRCA1 PDCD1 BRAF KRAS RET
CDKN2A PTEN DDR2 MET NTRK3
CTLA4 RRM1 EGFR NRAS NTRK1
4 ● ERCC1 TDP1 ERBB2 PIK3CA NRG1
ESR1 TERT ERBB4 PTEN FGFR1
FGFR1, FGFR2 TLE3 FBXW7 SMAD4 FGFR2
IFNGR TOP1 FGFR1 STK11 MBIP
ISG15 TUBB3 FGFR2 TP53 PDGFRA
The panel covers 107 recurrent gene fusions, 3′/5′ imbalance targets, MET AR-13324 14 skipping, 23 mRNA expression markers, and 55 DNA mutation region of interest regions in 20 genes relevant to NSCLC.
published methods [25–27]. Development and validation of the RNA NGS panel are described extensively in previously published work . The panel includes coverage of all DNA and RNA markers recommended by the NCCN NSCLC guideline  as part of broad molecular testing, in addition to emerging markers of clinical research value . RNA primer designs were selected to span exon-exon breakpoints specific to the target transcript. Additional designs enable detection of 3′/5′ imbalance of recurrently 3′ fused genes to support confirmation of known and detection of novel rearrangements. The RNA pool enables detection of MET exon 14 skipping and quantification of 23 other mRNA targets of published prognostic and theranostic value, including markers relevant to immune checkpoint inhibitor response such as PD-L1, PD-L2, IFNG, and CTLA4. Endogenous control targets utilized by the DNA- and RNA-specific real-time qPCR QC assays were selected based on an analysis of TCGA LUAD and lung squamous cell carcinoma (LUSC) cohorts to identify genes that are copy number neutral and stably expressed in diseased and normal lung tissue [31,32]. A set of three stably expressed reference genes, including the RT-qPCR controls, was included in the RNA pool to enable normalization of expression levels and provide quality control measures.
Specimen Preparation and Characterization
For each tissue specimen, multiple sections were scraped from the glass slides and combined into a single tube. The tissue was then incubated with xylene to remove residual paraffin followed by extraction using QIAamp® DNA FFPE Tissue Kit (QIAGEN, Germantown, MD) following manufacturer's protocol with the exclusion of RNase treatment in order to recover total nucleic acid (TNA). TNA was processed and eluted into 50 μl of ATE buffer (QIAGEN). FNA smears were isolated using the QIAamp® DNA FFPE Tissue Kit following manufacturer's protocol, excluding RNase digestion. TNA was eluted in 30 μl of nuclease-free water. Columns were eluted a second time with 50 μl into a fresh tube to capture any material that was not eluted in the first elution. TNA elutions were assessed for DNA and RNA yield using qPCR assays that quantify discrete populations of amplifiable DNA and RNA, QuantideX® qPCR DNA QC Assay (Asuragen, Inc., TX) and the qPCR RNA QC Assay (QuantideX® NGS RNA Lung Cancer Kit, Asuragen), respectively, according to manufacturer's protocol.
Library Preparation and Sequencing
Preanalytical QC of TNA was utilized to inform library input and downstream analysis of NGS results. TNA samples were stratified