title: Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls.
abstract: OBJECTIVE
  Cell free tumor DNA (cfDNA) circulating in blood has a great potential as biomarker for cancer clinical management. The objective of this study is to evaluate if cfDNA in blood plasma is detectable in early stage lung cancer patients.
  
  
  METHODS
  We extracted cfDNAs and tumor tissue DNAs from 8 lung adenocarcinoma patients. We also extracted cfDNAs from 8 normal controls. To evaluate copy number variations (CNV) and identify potential mutations, we performed low pass whole genome sequencing and targeted sequencing of 50 cancer genes. To accurately reflect the tumor-associated genomic abnormality burden in plasma, we developed a new scoring algorithm, plasma genomic abnormality (PGA) score, by summarizing absolute log2 ratios in most variable genomic regions. We performed digital PCR and allele-specific PCR to validate mutations detected by targeted sequencing.
  
  
  CONCLUSIONS
  The median yield of cfDNA in 400 ul plasma was 4.9 ng (range 2.25-26.98 ng) in patients and 2.32 ng (range 1.30-2.81 ng) in controls (p=0.003). The whole genome sequencing generated approximately 20 million mappable sequence reads per subject and 5303 read counts per 1Mb genomic region. Log2 ratio-based CNV analysis showed significant chromosomal abnormality in cancer tissue DNAs and subtle but detectable differences in cfDNAs between patients and controls. Genomic abnormality analysis showed that median PGA score was 9.28 (7.38-11.08) in the 8 controls and 19.50 (5.89-64.47) in the 8 patients (p=0.01). Targeted deep sequencing in tumor tissues derived from the 8 patients identified 14 mutations in 12 different genes. The PCR-based assay confirmed 3 of 6 selected mutations in cfDNAs. These results demonstrated that the PGA score and cfDNA mutational analysis could be useful tool for the early detection of lung cancer. These blood-based genomic and genetic assays are noninvasive and may sensitively distinguish early stage disease when combined with other existing screening strategies including low-dose CT scanning.
authors.affiliation: Department of Oncology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: xiashutj@gmail.com.
authors.forename: Shu
authors.initials: S
authors.lastname: Xia
authors.affiliation: Department of Biostatistics, University of Wisconsin, Milwaukee, WI 53201, USA. Electronic address: huangcc@uwm.edu.
authors.forename: Chiang-Ching
authors.initials: CC
authors.lastname: Huang
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: mle@mcw.edu.
authors.forename: Min
authors.initials: M
authors.lastname: Le
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: rdittmar@mcw.edu.
authors.forename: Rachel
authors.initials: R
authors.lastname: Dittmar
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: mdu@mcw.edu.
authors.forename: Meijun
authors.initials: M
authors.lastname: Du
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: tyuan@mcw.edu.
authors.forename: Tiezheng
authors.initials: T
authors.lastname: Yuan
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: gyongchen@mcw.edu.
authors.forename: Yongchen
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: yuwang@mcw.edu.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biostatistics, University of Wisconsin, Milwaukee, WI 53201, USA. Electronic address: xuexia@uwm.edu.
authors.forename: Xuexia
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: stsai@mcw.edu.
authors.forename: Susan
authors.initials: S
authors.lastname: Tsai
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: ssuster@mcw.edu.
authors.forename: Saul
authors.initials: S
authors.lastname: Suster
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: amackinnon@mcw.edu.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Mackinnon
authors.affiliation: Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: liwang@mcw.edu.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
mesh_terms: D000230:Adenocarcinoma / Q000097:blood* / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D016022:Case-Control Studies; D056915:DNA Copy Number Variations / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics*; D005260:Female; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood* / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D010950:Plasma Cells / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
keywords: Allele-specific PCR; Copy number variations; Digital PCR; Next generation sequencing; Non-small cell lung cancer; Plasma; cfDNA