Genomewide Copy Number Alteration Screening of Circulating Plasma DNA

Potential for the Detection of Incipient Tumors

L. Lenaerts; P. Vandenberghe; N. Brison; H. Che; M. Neofytou; M. Verheecke; L. Leemans; C. Maggen; B. Dewaele; L. Dehaspe; S. Vanderschueren; D. Dierickx; V. Vandecaveye; F. Amant; J. R. Vermeesch


Ann Oncol. 2019;30(1):85-95. 

In This Article

Patients and Methods


Aged people, 64 years and older, were recruited via senior societies (76% of participants) or via University Hospitals Leuven (Departments of Internal Medicine or Anaesthesiology, 24% of participants). Participants with a cancer history or reporting an early diagnosis were excluded. Peripheral blood (8–9 ml) was sampled in Streck tubes (Streck, Omaha). When a deviating GIPseq profile was detected, a second blood sample was taken for independent confirmation. Samples were collected between October 2015 and November 2017. The study was approved by the ethics committee of University Hospitals Leuven (S/55513). Written informed consent was obtained from all participants.

Cell-free Plasma and Genomic DNA Extraction

Plasma was isolated through a standard, two-step centrifugation procedure. cfDNA was extracted from 2 to 4 ml plasma, using, respectively, the QIAamp circulating nucleic acid Kit (Qiagen, Hilden, Germany; manual extraction) or the Maxwell HT ccfDNA kit (Promega, Madison; automated procedure). Median cfDNA concentration in plasma was 5.5 ± 0.5 ng/ml (median ± SEM). Genomic DNA from blood cells or formalin-fixed paraffin-embedded (FFPE) tumor biopsies was extracted after macrodissection using the Qiagen Blood and Tissue kit. Following sonication (Covaris M220), samples were electrophoretically run on the Agilent 2100 Bioanalyzer system to verify fragmentation. The target DNA fragment size for library preparation was 150–200 bp.

Genomewide Imbalance Profile Sequencing

DNA sequencing libraries were prepared using the TruSeq DNA Nano kit or Illumina ChipSeq kit (Illumina, San Diego). Whole-genome sequencing was carried out on a HiSeq2500 or 4000 (Illumina) using a V4 flowcell generating single-end 36 or 50 bp reads. Mean netto read count per sample was 9.6 ×10[6] reads. For Genomewide Imbalance Profiling (GIPseq), our previously described bioinformatics pipeline was applied, using genomewide parameters (quality score, QS), chromosomal parameters (z- and zz-score) and subchromosomal parameters.[7] QS was calculated as the standard deviation of the z-scores of all autosomes excluding the chromosomes with highest and lowest z-scores. A GIPseq profile was scored 'normal' when QS < 2 and no significant gains or losses were present across one of the chromosomes (i.e. |z-score| < 3.0 and |zz-score| < 3.0). The GIPseq profile was called 'aberrant' when QS<2, or when QS < 2 and one or more individual chromosomes were having |z-score| ≥ 3.0 and |zz-score| ≥ 3.0. Constitutional copy number variations were filtered out and GIPseq profiles were assessed by two independent researchers for their relevance as cancer-related CNAs, consulting online databases of chromosome aberrations in oncology.[8,9]

Array Comparative Genomic Hybridization and Fluorescent in Situ Hybridization

Array comparative genomic hybridization (aCGH) analysis was done as before,[10] using the 4×180K CytoSure Syndrome Plus Leuven Design microarray or the 8 ×60K CytoSure ISCA v2 microarray and analyzed with the CytoSure Interpret Software (OGT, Oxford, UK). Fluorescent in situ hybridization (FISH) was carried out according to standard procedures. DNA probes are described in supplementary Table S1, available at Annals of Oncology online.

Peripheral Blood Analyses

Analysis of hematological parameters (including cell counts and cytology), clinical biochemistry and protein electrophoresis of peripheral blood was done at the Laboratory Medicine of University Hospitals Leuven following standard procedures.

Whole-body MRI Screening

When two sequential plasma samples independently confirmed the presence of an aberrant GIPseq profile, subjects were sent for 3 Tesla whole-body diffusion Weighted MRI (WB-DWI MRI) imaging to be screened for the presence of cancer-like lesions, as described before.[11] We choose WB-DWI MRI due to its absent ionizing radiation, in-house experience, and similar diagnostic performance compared with PET/CT for detecting primary and metastatic malignancies.[5,12] As WB-DWI MRI was designed for diagnostic purposes rather than for screening purpose and from ethical considerations, we applied a low threshold for detection of possible tumor and incidental findings favoring high sensitivity for lesion detection over specificity. Detected lesions were examined via dedicated investigations in a second stage. When no lesions, indicative of malignancy, were observed, the participant was referred for two additional WB-DWI MRIs with a 3-month time interval.

Statistical Analysis

Two-tailed t-testing, assuming equal variances in unpaired samples was used to compare plasma cfDNA concentrations among study subgroups. Fisher's exact test was applied to determine the association between the frequency of chromosomal aberrations in plasma cfDNA and the age of the index.