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. 2017 Apr 4:7:120-126.
doi: 10.1016/j.bbacli.2017.03.006. eCollection 2017 Jun.

Detection of copy number alterations in cell-free tumor DNA from plasma

Olga Østrup  1 , Lise Barlebo Ahlborn  1   2 , Ulrik Lassen  2 , Morten Mau-Sørensen  2 , Finn Cilius Nielsen  1
Affiliations

Detection of copy number alterations in cell-free tumor DNA from plasma

Olga Østrup et al. BBA Clin. .

Abstract

Background: Somatic copy number alterations (SCNAs) occurring in tumors can provide information about tumor classification, patient's outcome or treatment targets. Liquid biopsies, incl. plasma samples containing circulating cell-free tumor DNA (ccfDNA) can be used to assess SCNAs for clinical purposes, however specify and reliability of methods have to be tested.

Methods: SNP microarrays (Affymetrix) were used to generate whole-genome copy number profiles from plasma ccfDNA (OncoScan) and paired tumor biopsies (CytoScan) from ten patients with metastatic cancers. Numerical, segmental and focal SCNAs were assessed using ASCAT/TuScan and SNP-FASST2.

Results: Aberrations in ccfDNA in 4 patients resembled numerical (76%) and segmental (80%) aberrations in tDNA. Three patients represented low correlation due to postponed sampling time, ccfDNA quality and possible treatment interference. Breakpoints of high-amplitude amplification were assessed with high accuracy and relative breakpoints difference of only 7% (0.02-37%). Similarly, biallelic losses were reliably detected. Array was 100% successful in detection of SCNAs on clinically relevant genes compared to SCNAs in tumor biopsies. Tracking of SCNAs changes during the treatment course of one patient also indicated that apoptosis/necrosis of non-cancerous cells presumably induced by treatment can influence ccfDNA composition and introduce false-negative findings into the analysis of liquid biopsies.

Conclusions: Genomic alterations detected in ccfDNA from liquid biopsies by comprehensive SNP array are reliable source for information for stratification of patients for targeted treatment.

General significance: Clinically relevant SCNAs can be detected in ccfDNA with high resolution and can therefore serve as an alternative to tumor biopsy in defining treatment targets.

Keywords: Array profiling; Circulating cell-free tumor DNA; Copy number alterations; Diagnostics.

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Figures

Fig. 1.
Fig. 1
Comparison of genomic profiles of ccfDNA and corresponding tDNA. (A) Overview plots showing probe densities on chromosomes for patient's paired samples. Upper plot for each patient represents probe densities distribution in ccfDNA and below probe densities in tDNA. Numbers above the plot indicates chromosome numbers. (B) Boxplots for percentages of genome subjected to aberrations in 7 patients with known genomic profile as assigned in ccfDNA by TuScan, and in tDNA assigned by ASCAT and SNPFASST2. (C) Plot showing percentual overlap of numerical (light-grey) and segmental (dark-grey) aberrations between tDNA and ccfDNA in individual patients. (D) Graph representing length of high amplitude amplifications detected in ccfDNA (light-grey) and in corresponding tDNA (dark-grey). “P” on x-axis indicates patient's number and “c” chromosome. Error bars on ccfDNA columns correspond to differences between individual break-points between tDNA and ccfDNA. (E) Chromosome view (NEXUS, BioDiscovery) on chromosome 9 in tDNA and ccfDNA. Arrows mark biallelic deletion in 9p21.3 involving CDKN2A/B and is enlarged is section to the right. (F) Aberrations detected on clinically relevant cancer genes in tDNA and ccfDNA. Dark-blue indicates alterations found in ccfDNA and tDNA. Light-grey marks alterations detected only in tDNA or only in ccfDNA. Light-blue marks corresponding alterations after performance of manual correction.
Fig. 2.
Fig. 2
Patient case. (A) Longitudinal study of genomic aberrations in tDNA and ccfDNA obtained from Patient 5 before and during treatment. The top part of the figure shows whole genome profiles of tDNA, analyzed by CytoScan, originating from diagnostic (blue rectangle), pre- (green rectangle) and in-treatment (red rectangle) biopsies. Below are whole genome profiles of ccfDNA analyzed by OncoScan, and sampled after the initiation of treatment 1 and 2 (blue empty rectangle), between treatments 2 and 3 (green empty rectangle) and after 73 days of treatment 3 (red empty rectangle). Notice the absence of aberrations in cfDNA obtained under treatment. (B) Distribution of numerical and segmental aberrations and of focal alterations in cancer related genes in diagnostic, pre-treatment and in-treatment biopsies and chronologically placed ccfDNA obtained between treatments 2 and 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. S1.
Fig. S1
Titration experiment. Whole genome views on tDNA analyzed on CytoScan arrays with recommended concentration and analyzed on OncoScan arrays with total input of 25, 10 and 5 ng. Table shows QC parameters of samples analyzed on OncoScan.
Fig. S2.
Fig. S2
Examples of high-amplitude amplification. Examples of high amplitude amplifications detected in tumor DNA and in corresponding ccfDNA occurring in patient 1 (amplification of ERBB2) and in patient 2 (large amplification including e.g. MET).
Fig. S3.
Fig. S3
Examples of biological bias in ccfDNA analysis. Whole genome views of tDNA and ccfDNA of Patient 5 and Patient 7. Patient 5 displays a less altered genomic profile in ccfDNA collected 95 days before the tumor biopsy. Patient 7, exposed to regorafenib treatment under sampling of ccfDNA, shows ccfDNA genomic profile without aberrations, i.e. corresponding to genomic profile of normal cells
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