Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 31;10(1):42.
doi: 10.1186/s40478-022-01335-6.

Droplet digital PCR-based analyses for robust, rapid, and sensitive molecular diagnostics of gliomas

Marietta Wolter  1 , Jörg Felsberg  1 , Bastian Malzkorn  1 , Kerstin Kaulich  1 , Guido Reifenberger  2   3
Affiliations

Droplet digital PCR-based analyses for robust, rapid, and sensitive molecular diagnostics of gliomas

Marietta Wolter et al. Acta Neuropathol Commun. .

Abstract

Classification of gliomas involves the combination of histological features with molecular biomarkers to establish an integrated histomolecular diagnosis. Here, we report on the application and validation of a set of molecular assays for glioma diagnostics based on digital PCR technology using the QX200™ Droplet Digital™ PCR (ddPCR) system. The investigated ddPCR-based assays enable the detection of diagnostically relevant glioma-associated mutations in the IDH1, IDH2, H3-3A, BRAF, and PRKCA genes, as well as in the TERT promoter. In addition, ddPCR-based assays assessing diagnostically relevant copy number alterations were studied, including 1p/19q codeletion, gain of chromosome 7 and loss of chromosome 10 (+ 7/-10), EGFR amplification, duplication of the BRAF locus, and CDKN2A homozygous deletion. Results obtained by ddPCR were validated by other methods, including immunohistochemistry, Sanger sequencing, pyrosequencing, microsatellite analyses for loss of heterozygosity, as well as real-time PCR- or microarray-based copy number assays. Particular strengths of the ddPCR approach are (1) its high analytical sensitivity allowing for reliable detection of mutations even with low mutant allele frequencies, (2) its quantitative determination of mutant allele frequencies and copy number changes, and (3) its rapid generation of results within a single day. Thus, in line with other recent studies our findings support ddPCR analysis as a valuable approach for molecular glioma diagnostics in a fast, quantitative and highly sensitive manner.

Keywords: DNA copy number variation; Droplet digital PCR; Glioma; Molecular diagnostics; Mutation; Single nucleotide variation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Detection of TERT promoter mutations in FFPE DNA extracted from gliomas using ddPCR. a Fluorescent intensity of the droplets after amplification of a 113 bp-fragment of the TERT promoter (TERTp) region using the ddPCR expert design assays (Bio-Rad Laboratories). The individual lanes correspond to: 1, TERTp “C228T” mutation control DNA; 2, TERTp “C250T” mutation control DNA; 3, TERTp wildtype control DNA; 4, no template control; 5, glioblastoma, IDH-wildtyp, CNS WHO grade 4, and 6, oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, both with TERTp “C228T” and “C250T” mutations. X axis, number of droplets with fluorescence; Y axis, fluorescence intensity detected in the FAM-channel (blue dots) and HEX-channel (green dots); pink line, threshold; grey dots, droplets with background fluorescence of non-incorporated probes. b Validation of the two TERT promoter mutations in the IDH-wildtype glioblastoma using Sanger sequencing. Red arrow heads pointing to the “C250T “ (left) and “C228T” (right) mutation. The numbering of the samples corresponds to a. c Mutant allele frequency (MAF) was measured by ddPCR using different amounts of input FFPE DNA generated by serial dilution of samples with either a TERTp “C228T” (mean MAF 37.3%) or a “C250T” (mean MAF 38.1%) mutation with distilled water. d 25 ng of total input FFPE DNA was used for ddPCR. A TERTp “C228T” (mean MAF 34.7%) and a TERTp “C250T” DNA sample (mean MAF 31.7%) were mixed with wildtype DNA resulting in predefined templates with 50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, and 0.4% TERT promoter-mutant DNA in a TERT promoter-wildtype background
Fig. 2
Fig. 2
Loss of heterozygosity (LOH) on chromosomal arms 1p and 19q detected by ddPCR-based SNP analysis. a Patterns of ddPCR-based SNP analyses for LOH on 1p and 19q in 27 gliomas, previously shown to either have retained (tumor samples 1–5 and 27) or lost 1p and 19q (tumor samples 6–26). In ten patients with IDH-mutant and 1p/19q-codeleted oligodendrogliomas, matched DNA samples extracted from tumor tissue (T) and blood leucocytes (B) were investigated. In the remaining 17 patients, only T DNA samples were investigated. The cases 1–5 without 1p/19q codeletion were used to establish the cut-off value for the detection of LOH. Note that all patients showed one or more informative (heterozygous) SNPs on 1p, while 3 of the 27 patients lacked an informative SNP on 19q. Presence or absence of 1p/19q-codeletion in each individual case was validated by an independent method. White rectangles, non-informative SNP; light grey rectangles, informative SNP with retained heterozygosity; black rectangles, informative SNP with loss of heterozygosity; crossed rectangles, data not evaluable. b Exemplary presentation of a two-dimensional plot generated by the Quantasoft™ Software (Bio-Rad Laboratories). Shown are the results of the ddPCR-based analysis of SNP rs1493695 in case 9, who exhibited a LOH at this locus. X axis, fluorescence intensity detected in the HEX-channel (channel 2); Y axis, fluorescence intensity detected in the FAM-channel (channel 1); pink line, threshold; grey dots, droplets with background fluorescence; green dots, droplets with fluorescence detected in the HEX-channel; blue dots, droplets with fluorescence detected in the FAM-channel; orange dots, droplets with signals in both channels. The tumor-DNA showed a reduced number of droplets in the FAM-channel (arrow) compared to the number of droplets in the HEX-channel (green dots). The blood-derived DNA of this patient is heterozygous for this SNP with nearly the same number of droplets counted in the FAM- and HEX-channel. c Results of ddPCR analyses of additional SNPs located on 1p or on 19q in 12 selected cases, including 10 tumors with only one informative locus on one or both chromosome arms and two of the cases that were not informative at the five initially studied SNPs on 19q (see Fig. a above). Note that all cases demonstrated additional informative SNPs that confirmed the initial results and allowed for complete 1ß/19q copy number evaluation in the two cases that initially were not informative on 19q
Fig. 3
Fig. 3
Patterns of ddPCR-based SNP analyses for loss of heterozygosity on chromosome 10 in 13 control blood samples and 22 selected tumor samples, previously demonstrated to have losses on 10q only (tumor samples 1- 3), no losses on 10 (tumor sample 4), or losses on both 10p and 10q (tumor samples 5–22). White rectangles, non-informative SNP; light grey rectangles, informative SNP with retained heterozygosity; black rectangles, informative SNP with loss of heterozygosity. Tumor sample 1, astrocytoma, IDH-mutant, CNS WHO grade 2; Tumor sample 2, astrocytoma, IDH-mutant, CNS WHO grade 4; Tumor samples 3–22, glioblastoma, IDH-wildtype, CNS WHO grade 4. The majority of samples showed at least one informative (heterozygous) SNP on each chromosomal arm, except for 3 samples that were not informative at the SNPs on 10p, one sample that was not informative at the SNPs on 10q, and one sample that was not informative at the 8 SNPs at both chromosomal arms. Presence or absence chromosome 10 losses in the individual tumor cases were independently validated by other methods (see text)
Fig. 4
Fig. 4
Gene copy number changes on chromosome 7 detected by ddPCR-based copy number analysis in 10 control blood samples and 20 tumor samples, previously shown to have a chromosome 7 gain (tumor samples 1–13) or a balanced chromosome 7 status (tumor samples 14–20). The threshold for a copy number gain at an individual genomic locus was set to ≥ 2.5 according to Crespo et al. [43]. Tumor samples 1–15, glioblastoma IDH-wildtype, CNS WHO grade 4; Tumor sample 16, astrocytoma, IDH-mutated, CNS WHO grade 4; Tumor samples 17–18, diffuse midline glioma, H3 K27-altered; Tumor sample 19, pilocytic astrocytoma, CNS WHO grade 1; Tumor sample 20, others (atypical teratoid/rhabdoid tumor). White rectangles, copy number value < 2.5; light grey rectangles, copy number value ≥ 2.5 and < 5.0 (low-level copy number gain); black rectangles, copy number value ≥ 5.0 (high-level copy number gain/gene amplification); crossed rectangle, data not available. None of the control blood samples displayed evidence of gains of whole chromosome 7 indicative of trisomy 7. The results obtained by ddPCR for chromosome 7 gain by ddPCR were independently validated by other methods. Note that individual tumors demonstrated evidence for focal high-level copy number changes on the background of whole chromosome 7 gain
Fig. 5
Fig. 5
Detection of focal copy number changes of the EGFR gene on chromosome 7p11.2. Copy number analysis was done by ddPCR using a PrimePCR™ ddPCR copy number assay (Bio-Rad Laboratories) for the analysis of exon 7 and a newly designed ddPCR assay for exon 28 copy number changes. a Schematic representation of EGFR copy number changes. Upper three rows, Results of EGFR amplification analysis by ddPCR assays. Lower two rows, Data obtained by independent methods. Tumor cohort 1 corresponds to selected cases with known EGFR copy number status before ddPCR analysis for EGFR amplification. In tumor cohort 2, results obtained by ddPCR were validated afterwards by other methods. Tumor samples 1–15, 26–44, glioblastoma, IDH-wildtype, CNS WHO grade 4 (samples 1 and 2, 11 and 12 as well as 13 and 14 are pairs of primary and recurrent tumor); Tumor samples 16–19, oligodendroglioma, IDH-mutant, and 1p/19q codeleted, CNS WHO grade 2; Tumor samples 20–25, oligodendroglioma, IDH-mutant, and 1p/19q codeleted, CNS WHO grade 3. White rectangles, no EGFR amplification (EGFR copy number < 5.0); black rectangles, EGFR gene amplification (EGFR copy number ≥ 5.0); grey rectangles; EGFRvIII variant; crossed rectangles, data not available. b Exemplary presentation of two-dimensional plots generated by the Quantasoft™ Software. Shown are the results of the ddPCR-based analysis of EGFR exon 7 and exon 28 copy number changes in the tumor samples 6 and 18. X axis, fluorescence intensity detected in the HEX-channel (channel 2); Y axis, fluorescence intensity detected in the FAM-channel (channel 1); pink line, threshold; grey dots, droplets with background fluorescence; green dots, droplets with fluorescence detected in the HEX-channel; blue dots, droplets with fluorescence detected in the FAM-channel; orange dots, droplets with signals in both channels. Tumor sample 6 exhibited a high-level EGFR amplification and the EGFRvIII variant. Note the high number of blue dots in channel 1 for EGFR exon 28 compared to the green dots in channel 2 for the reference gene, whereas the number of blue dots in channel 1 for exon 7 are markedly lower than for exon 28. Tumor sample 18 showed no EGFR copy number change with nearly the same number of droplets for exon 7 and exon 28 in both channels
Fig. 6
Fig. 6
Detection of CDKN2A tumor suppressor gene deletions in gliomas by ddPCR-based copy number analysis. a Results of Pearson´s correlation analysis of the calculated CDKN2A copy number compared to the detected copy number (CN) in mixtures of U87-MG DNA, which has a homozygous deletion of CDKN2A, and a control DNA that retained both CDKN2A copies. The copy numbers of the undiluted DNA were determined by ddPCR (100% U87-MG DNA: mean CN 0.0; control-DNA: mean CN 1.85 ± 0.02). b Results of CDKN2A copy number analyses using a PrimePCR™ ddPCR copy number assay (Bio-Rad Laboratories) in 66 glioma samples in comparison to other methods (NGS or qPCR). homodel, homozygous deletion; hemidel, hemizygous deletion; ddPCR, droplet digital PCR; NGS, gene panel next generation sequencing [20]; qPCR, semiquantitative real-time PCR. Note that ddPCR-based analysis identified three tumors with homozygous CDKN2A deletion that were considered as showing hemizygous deletions with the other methods. c Fluorescent intensity of the droplets after duplex-PCR using a PrimePCR™ ddPCR copy number assay (Bio-Rad Laboratories) for amplification of a 66 bp-fragment of the CDKN2A locus (upper row) together with self-designed primers for amplification of a 86 bp-reference gene locus (lower row) (NCKAP5, see Additional file 1: Table S2). X axis, number of droplets with fluorescence; Y axis, fluorescence intensity detected in the FAM-channel (Channel 1, blue dots) and HEX-channel (Channel 2, green dots); pink line, threshold; grey dots, dots with background fluorescence of non-incorporated probes; Lane 1, the IDH-mutated astrocytoma, CNS WHO grade 4, exhibited a homozygous deletion of the CDKN2A locus (CN < 0.5). Note that the number of blue droplets are markedly lower than the number of green droplets indicating a homozygous CDKN2A deletion. The few remaining blue droplets were caused by CDKN2A non-deleted cells. Lane 2, the glioblastoma, IDH-wildtype, CNS WHO grade 4, retained both copies of CDKN2A and showed nearly the same numbers of droplets in channel 1 and channel 2. Lane 3, no template control
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Louis DN, Perry A, Wesseling P, et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 2021;23:1231–1251. doi: 10.1093/neuonc/noab106. - DOI - PMC - PubMed
    1. Louis DN, Wesseling P, Aldape K, et al. cIMPACT-NOW update 6: new entity and diagnostic principle recommendations of the cIMPACT-Utrecht meeting on future CNS tumor classification and grading. Brain Pathol. 2020;30:844–856. doi: 10.1111/bpa.12832. - DOI - PMC - PubMed
    1. Brat DJ, Aldape K, Colman H, et al. cIMPACT-NOW update 5: recommended grading criteria and terminologies for IDH-mutant astrocytomas. Acta Neuropathol. 2020;139:603–608. doi: 10.1007/s00401-020-02127-9. - DOI - PMC - PubMed
    1. Leske H, Dalgleish R, Lazar AJ, et al. A common classification framework for histone sequence alterations in tumours: an expert consensus proposal. J Pathol. 2021;254:109–120. doi: 10.1002/path.5666. - DOI - PubMed
    1. Louis DN, Perry A, Reifenberger G, et al. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 2016;131:803–820. doi: 10.1007/s00401-016-1545-1. - DOI - PubMed

Publication types

Substances

-