Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter-and intra-assay as well as intra-and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity

DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms

Epac 1 and Epac 2 (Epac1/2; exchange factors directly activated by cAMP) are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms (Epac2A, Epac2B and Epac2C) that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the Epac2 locus that are essential for its transcriptional control

DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms

Epac 1 and Epac 2 (Epac1/2; exchange factors directly activated by cAMP) are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms (Epac2A, Epac2B and Epac2C) that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the Epac2 locus that are essential for its transcriptional control

The Epac2B-promoter is hypomethylated in the adrenal gland.

<p>Bisulfite sequencing analyses of the <i>Epac2</i> B-promoter (containing 6 CpG sites across a region of 438 bp, spanning nucleotides 72054618–72055055 of chr2). Genomic DNA was prepared from adrenal cortex (A; 8 mice), brain (B; 7 mice), pituitary (C; 5 mice), liver (D; 7 mice), freshly isolated hepatocytes (E; 3 mice), endocrine pancreas (F; 5 mice), exocrine pancreas (G; 5 mice) and kidney (H; 7 mice). Bisulfite sequencing was performed on pooled DNA from the number of mice indicated above. The overall percentage of demethylated CpG sites and the number of clones analyzed were (number of clones in parentheses): 94% (39) in (A), 20% (46) in (B), 7% (28) in (C), 19% (46) in (D), 13% (58) in (E), 9% (38) in (F), 8% (12) in (G) and 23% (41) in (H). Each horizontal line represents one analyzed clone. Black circles: methylated CpG sites, open circles; demethylated CpG sites. Representative clones are shown for A-F and H.</p

The predicted promoters regions of <i>Epac2</i> contains CpG dinucleotides and hold transcriptional activity.

<p><b>A)</b> Schematic drawing of the predicted promoter regions of <i>Epac2</i>. The promoter region directing expression of Epac2A (chr2 71980880–71981553; upper panel) contains a potential INR- (initiator) like element, a TATA-box and two putative TSSs. A CGI with 51 CpG sites (indicated by grey lollipops) covers the promoter region. The promoter region directing expression of Epac2B (chr2 72054237–72054766; middle panel) contains a putative INR element and a M3 element. This region contains 6 CpG sites as indicated by lollipops. The promoter region directing expression of Epac2C (72179738–72180207; lower panel) contains three alternative TSSs (TSS1–3), as well as an INR element as also described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Ueno1" target="_blank">[6]</a>. Transcription is initiated from TSS1/2 and not TSS3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Ueno1" target="_blank">[6]</a>. This region contains 13 CpG sites. The translational start sites are indicated by bent arrows. To identify novel potential promoter regions in <i>Epac2</i> the sequences corresponding to potential promoter regions were analyzed by the FlyBase eukaryotic promoter prediction computational tool (<a href="http://flybase.org" target="_blank">http://flybase.org</a>) and rVISTA evolutionary conservation analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Loots1" target="_blank">[17]</a>. The promoter regions shown correspond to the genomic regions that were analyzed by bisulfite sequencing in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-g004" target="_blank">figures 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-g006" target="_blank">6</a>. Note that the CpG-site marked with a “Z” (upper panel) is included in primer sequence and therefore not included in the BSP analysis). <b>B)</b> The reporter gene plasmids containing the <i>Epac2</i> promoter regions (pCpG/prom-Epac2A, pCpG/prom-Epac2B and pCpG/prom-Epac2C; 200 ng), the positive control vector pCpG/CMV-EF1alpha (200 ng) and the pCpG-basic vector (containing no promoter/enhancer; 200 ng), were transfected into Cos-1 cells. Before transfection the plasmids were either <i>in vitro</i> methylated by the <i>Sss</i>I CpG methylase (black bars) or left untreated (grey bars). IVM; <i>in vitro</i> methylation. The luciferase activities are presented as average +/− stdev, n = 9.</p

The CGI of the Epac2A-promoter is demethylated in all tissues examined.

<p>Bisulfite sequencing analyses of the <i>Epac2</i> A-promoter (containing 50 CpG sites across a region of 441 bp, spanning nucleotides 71980937–71981377 of chr2). DNA was prepared from brain (A), pancreas (B), adrenal cortex (C), liver (D) and kidney (E). Bisulfite sequencing was performed on pooled DNA; from 7 mice in A–B and D–E, and from 8 mice in C. For all tissues, the percentage of methylated CpG sites was ≤1%. The total number of analyzed clones were: 32 in (A), 30 in (B), 12 in (C), 35 in (D) and 34 in (E). Each horizontal line represents one analyzed clone. Black circles: methylated CpG sites, open circles; demethylated CpG sites. Representative clones are shown for A–B and D–E.</p

The Epac2C-promoter is hypomethylated in liver.

<p>Bisulfite sequencing analyses of the <i>Epac2</i> C-promoter (containing 13 CpG sites across a region of 469 bp, spanning nucleotides 72179672–72180140 of chr2). Genomic DNA was prepared from liver (A; 7 mice), freshly isolated hepatocytes (B; 3 mice), hepatocytes cultured for 11 days (C; 3 mice), adrenal cortex (D; 8 mice), endocrine pancreas (E; 5 mice), brain (F; 7 mice), pituitary (G; 5 mice), exocrine pancreas (H; 5 mice) and kidney (I; 7 mice). Bisulfite sequencing was performed on pooled DNA from the number of mice indicated above. The overall percentage of demethylated CpG sites and the number of clones analyzed were (number of clones in parentheses): 78% (52) in (A), 95% (48) in (B), 99.7% (64) in (C), 7% (29) in (D), 12% (15) in (E), 10% (40) in (F), 4% (28) in (G) 12% (15) in (H) and 6% (31) in (I). Each horizontal line represents one analyzed clone. Black circles: methylated CpG sites, open circles; demethylated CpG sites. Representative clones are shown for A-D, F, G and I.</p

Epac2 protein isoforms and genomic organization of <i>Epac2</i>.

<p><b>A)</b><b> </b> Schematic illustration of the protein domains of the Epac2 isoforms. The regulatory domain consists of one (Epac2B and Epac2C) or two (Epac2A1/2A2) cAMP binding domains (cAMP-A and cAMP-B) and a dishvelled, Egl–10 and pleckstrin homology domain (DEP; except for Epac2C). The catalytic region consists of an exchange activity domain that catalyses Rap activation (RasGEF), a Ras-exchange motif (REM) and a Ras association (RA) domain. The full-length isoform (Epac2A1) consists of 1011 amino acids (aa), while Epac2A2 is deduced to contain 993 aa. Epac2A2 is identical to Epac2A1, except for an 18 aa deletion corresponding to exon 7. Epac2B consists of 867 aa, and Epac2C of 696 aa. <b>B)</b> Genomic organization and alternative promoters at the <i>Epac2</i> locus. Coding exons are numbered and illustrated by black boxes. White parts of exons indicate untranslated regions (UTRs). TSSs for the Epac2A, Epac2B and Epac2C isoforms are indicated by bent arrows, while the corresponding translational start sites (ATGs) are indicated by open arrowheads. The filled arrowhead in exon 31 indicates a shared stop codon. The figure is based on the NCBI reference sequence: NC_000068.6, Chr2∶71819344–72094433, and is expanded in 5′-region to include the CpG-island (start; nt 71818997) and in the 3′-region to include the UTR of exon 31 (end; nt 72095526). Note the different scales for exons and introns.</p

Expression of Epac2 isoforms.

<p><b>A)</b> mRNA was prepared from mouse brain, adrenal glands, liver, kidney and endocrine and exocrine pancreas, converted to cDNA, and RT-PCR was performed with primers as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-t001" target="_blank">table 1</a>. +/−; presence and absence of reverse transcriptase (RT). <b>B–C)</b> Immunoblotting with Epac2 antibodies was performed on protein extracts prepared from Cos1-cells overexpressing Flag-tagged Epac2A, Epac2B or Epac2C (20 μg; B, C; Cos1-cells do not express endogenous Epac2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Islam1" target="_blank">[9]</a>), and extracts prepared from mouse brain, adrenal gland, liver and kidney (250–300 μg; B) as well as from brain and endocrine and exocrine pancreas (250–300 μg; C). The migration of the different isoforms is indicated by arrows. Immunoblotting against actin is shown below each blot. The apparent absence of actin in the Cos-1 cell extracts in B is explained by a very short exposure time of the blot (as only 20 μg of Cos-1 extract was loaded on the gel compared to 200–250 μg of tissue extract).</p

PCR primers for construction of reporter plasmids (pCpG-constructs).

<p>PCR primers for construction of reporter plasmids (pCpG-constructs).</p