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. 2022 Mar 31;10:42. doi: 10.1186/s40478-022-01335-6

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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