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Review
. 2021 Apr 29;22(9):4732.
doi: 10.3390/ijms22094732.

Copy Number Variation and Rearrangements Assessment in Cancer: Comparison of Droplet Digital PCR with the Current Approaches

Vincenza Ylenia Cusenza  1 , Alessandra Bisagni  2 , Monia Rinaldini  3 , Chiara Cattani  3 , Raffaele Frazzi  1
Affiliations
Review

Copy Number Variation and Rearrangements Assessment in Cancer: Comparison of Droplet Digital PCR with the Current Approaches

Vincenza Ylenia Cusenza et al. Int J Mol Sci. .

Abstract

The cytogenetic and molecular assessment of deletions, amplifications and rearrangements are key aspects in the diagnosis and therapy of cancer. Not only the initial evaluation and classification of the disease, but also the follow-up of the tumor rely on these laboratory approaches. The therapeutic choice can be guided by the results of the laboratory testing. Genetic deletions and/or amplifications directly affect the susceptibility or the resistance to specific therapies. In an era of personalized medicine, the correct and reliable molecular characterization of the disease, also during the therapeutic path, acquires a pivotal role. Molecular assays like multiplex ligation-dependent probe amplification and droplet digital PCR represent exceptional tools for a sensitive and reliable detection of genetic alterations and deserve a role in molecular oncology. In this manuscript we provide a technical comparison of these two approaches with the golden standard represented by fluorescence in situ hybridization. We also describe some relevant targets currently evaluated with these techniques in solid and hematologic tumors.

Keywords: FISH; MLPA; copy number variation assessment; droplet digital PCR.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(A) Graphical representation of the FISH technique: from the adhesion of the nucleic acid to the hybridization of the probes. (B) On the left panels, example of a gene deleted in chronic lymphocytic leukemia. The slides show the TP53 gene (17p13.1) represented by the red spot [cosmid probes for TP53 deletion (Cytocell)] and the control (centromere 17) by the green spot. In the normal nucleus and metaphase, the number of red spots is equal to the number of green spots and they are both diploid. In the deleted nucleus the number of red spots is equal to 1, instead of two. On the right panels, example of translocation in chronic myeloid leukemia. The translocation in question concerns the t(9;22) of the genes ABL (9q34.12) and BCR (22q11.21), known as the Philadelphia chromosome. In the upper slide a normal karyotype is represented. BCR and ABL are clearly distinguishable and highlighted by a dual fusion cosmid probes for BCR/ABL (ABL1) translocation. The lower slide shows the translocation: the two spots overlap and are displayed as a yellow spot. (C) Example of rearranged and amplified genes: on the left ALK and MYC genes, respectively, in cervical cancer and B-cell lymphoma. The rearrangement in FISH can be seen because the two spots, one that identifies the gene (red tag; LSI ALK dual color break apart probe (Vysis) and LSI MYC dual color break apart rearrangement probe (Vysis)) and one that identifies the centromere, which is used as a control (green tag), are distant. On the right panel, example of amplification of the HER2 gene in breast cancer. The red spots (identified in the images by dual color probe LSI HER2 spectrum orange) that identify HER2 gene are in a greater number compared to the green spots that identify the control, represented by centromere 17 (CEP17 Spectrum Green PathVysion Vysis). DAPI staining (blue) is used for the nuclei morphology in all the presented panels. The magnification is 63×.
Figure 2
Figure 2
MLPA procedure. Reference DNA samples are required to determine the relative copy number of each of the probes. After denaturation of the target DNA, the left and right probe oligos (LPO and RPO respectively) are hybridized to their target sequence overnight (at least 16 h). The next day, the two oligonucleotides are enzymatically ligated and a barcode oligo (BO) is incorporated into the amplicon for sample identification. After PCR, products are separated by length. Finally quality check is performed and probe ratios calculated. A probe ratio of 1.0 corresponds to a normal diploid copy number, a probe ratio of 0.5 to a heterozygous deletion.
Figure 3
Figure 3
(A) Overview of the ddPCR procedure. (B) The graphs show examples of the analysis of TP53 CNV obtained from a previous work carried out by Frazzi and coauthors [43]. These graphs show the wild-type (wt) TP53 gene (right panel), TP53 with 50% deletion (middle panel) and 85% deletion (left panel). The increase in gene deletion corresponds to a decrease of the positive population (orange dots, red circled). The orange population is the double-positive one.
Figure 3
Figure 3
(A) Overview of the ddPCR procedure. (B) The graphs show examples of the analysis of TP53 CNV obtained from a previous work carried out by Frazzi and coauthors [43]. These graphs show the wild-type (wt) TP53 gene (right panel), TP53 with 50% deletion (middle panel) and 85% deletion (left panel). The increase in gene deletion corresponds to a decrease of the positive population (orange dots, red circled). The orange population is the double-positive one.
Figure 4
Figure 4
Representation of the advantages and disadvantages of the FISH, MLPA and ddPCR.
See this image and copyright information in PMC

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