Edited by: Parvin Mehdipour, Tehran University of Medical Sciences, Iran
Reviewed by: Pankaj Kumar Ahluwalia, Augusta University, United States; Umamaheswaran Gurusamy, University of California San Francisco, United States
*Correspondence: Julia M. A. Pickl,
This article was submitted to Cancer Genetics, a section of the journal Frontiers in Oncology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB analysis is required to maximize diagnostic yield for patients.
We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established.
We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches.
Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.
LB enables identification of genetic sequence variants in circulating free DNA (cfDNA) from plasma and allows stratification of patients that will benefit from targeted therapies. One example is the detection of oncogenic driver variants in plasma of non-small cell lung cancer (NSCLC) patients that is associated with response to tyrosine kinase inhibitor therapy (
LB is an alternative to standard tissue biopsy, and is increasingly applied when tissue accessibility is limited, in case tissue biopsy leads to insufficient quality or quantity of material, or the result of a tissue biopsy analysis is expected to take longer than a LB assay (
A major advantage of LB compared to tissue biopsy is that LB is able to cover the genetic heterogeneity of disease (
Aiming at offering LB to as many patients as possible, clinical laboratories must 1) cover the important therapy relevant variants, 2) use validated highly sensitive and accurate methods for detection of very low variant frequencies in plasma with well-defined cutoffs for variant reporting and quantification, and 3) offer both complementary and combined analysis of plasma and tissue biopsies for full flexibility and to maximize diagnostic yield.
The aim of this study is to develop aNGS-based assay that combines liquid and tissue biopsy (including tumor tissue and fibroblasts) to maximize flexibility for both patients and clinicians and to increase diagnostic yield at acceptable cost. We selected the most important therapy relevant variants and considered latest technological improvements for detection of very low frequency variants commonly present in plasma (
The study was approved by the ethics commission of the Bavarian Medical Association (No. 17059) and is registered with the German registry for clinical trials (trial registration ID: DRKS00012890). All participants or their legal guardian provided informed written consent prior to blood and tissue specimen collection. For the case report, the legal guardian provided written consent for publication of pictures. The study was performed in accordance with the Declaration of Helsinki.
Skin fibroblast and plasma samples were obtained in parallel from a ten-year old girl with clinically diagnosed asymmetric overgrowth syndrome. Another plasma sample was obtained from one tumor patient and FFPE tissue samples were collected from a total of 12 tumor patients (
Information on reference materials, DNA extraction, kit design, library preparation and sequencing, karyotyping, microarray analysis, and whole-exome sequencing (WES) are provided in the
Raw data (FASTQ.GZ format) was uploaded to the VARVIS® platform and aligned against the hg38 reference genome followed by variant calling using the bioinformatics pipeline VARFEED worker 1.5.1 with
To allow 95% confidence for detection of variants present with low VAFs, we aimed to analyze at least 60 low frequency variants with both LB and tissue analysis (
Validation of the tissue analysis was performed using the Quantitative Multiplex Reference Standard FFPE (Horizon), ten well-characterized clinical samples and the Ashkenazim Son FFPE Reference Standard NA24385 (SensID) as WT control. Further,
To limit the number of false positive (FP) variants, we established the LOB at 0.25% VAF based on the background noise at genomic positions expected to be WT in all samples as cutoff for variant calling. With a target region of 102 kb the number of positions in each of the validation samples for both LB and tissue analysis exceeds the required 60 WT positions for determination of the LOB (
For determination of the LOD we investigated the detection rate of 90 spike-in variants from 0.5% to 5% VAF for LB analysis and of 573 variants with 8% to 16% VAF for tissue analysis. The number of analyzed spike-in variants exceeds the required 60 variants for determination of the LOD with 95% confidence (
Variants were detected above the LOB of the respective analysis (LB: 0.25%, tissue: 5%) as cutoff for true-positive variant detection. The variants detected in processed samples were compared to the intersected trusted regions of the GIAB version 3.3.2 (
In addition to sensitivity and PPV, we also assessed trueness, precision and the total error of 30 spike-in variants with 0.5% VAF, 1% VAF and 5% VAF, respectively, to establish the LOQ (
Our aim was to develop a cost-efficient workflow for both LB and tissue analysis which covers the most important actionable genes and variants in solid tumors and mosaic diseases based on a custom hybrid-capture panel for targeted sequencing on the basis of European Society for Medical Oncology (ESMO) guidelines (
To allow maximal flexibility we aimed at enabling plasma and tissue sample processing either in parallel or independently (
Combined analysis workflow for plasma and tissue samples. Created with
The Duplex tags added during library preparation allow the bioinformatics identification of all amplified DNA fragments originating from a single strand of the original DNA molecule (
Duplex Consensus building.
With our approach, we achieved a mean effective coverage of 1,735x and 1,496x for plasma and tissue samples, respectively. For plasma samples in median ~98.6% of regions were covered with >250x, and for tissue samples ~100% of regions were covered with >100x (
We assessed the detection rate in plasma samples using cfDNA isolated from Seraseq® ctDNA Complete™ Reference Materials (VAF 0%-5%) (Seracare). The Seraseq® ctDNA Complete™ Reference Material includes 40 clinically relevant variants across 28 genes at 0.5%, 1% and 5% and a WT control sample. Of these variants 15 (8 SNVs and 7 InDels) are located in the kit target region. Only variants with a VAF above the LOB of 0.25% for the LB Duplex workflow that are present in at least eight consensus reads were called. Using these parameters, we observed a SNV detection rate of 100% at 0.5%, 1.0% and 5.0% VAF and an InDel detection rate of 79% at 0.5% VAF (average of duplicate measurement), and of 100% at 1% and 5% VAF, with highly similar detection in forward and reverse reads (
Detection rate of plasma analysis.
We further analyzed the detection rate in tissue samples using gDNA isolated from Quantitative Multiplex Reference Standard FFPE (Horizon), ten well-characterized clinical samples and the Ashkenazim Son FFPE Reference Standard NA24385 (SensID) as WT control. 7/11 clinically relevant variants of the Quantitative Multiplex Reference Standard FFPE (Horizon) and 11/11 clinically relevant variants in ten clinical samples were covered in the kit target region and above the LOB of 5% for the tissue analysis workflow. For tissue samples, a detection rate of 100% was observed since all 18 variants present in different reference materials with VAFs above the LOB of 5% were detected. As expected, no clinically relevant variants were detected in the Ashkenazim Son FFPE Reference Standard NA24385 (SensID) WT control (
To determine the sensitivity of LB analysis, we considered TP in all reference materials with 0.5%, 1.0% and 5.0% VAF. In total, 48/48 SNVs and 39/42 InDels were detected, resulting in 100% and 92.9% sensitivity for SNVs and InDels, respectively (
Sensitivity and PPV of LB and tissue analyses.
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PPV and Sensitivity are calculated based on TP, FN, and FP results.
TP, true positives; FN, false negatives; FP, false positives; PPV, positive predictive value.
To evaluate sensitivity and PPV of the tissue analysis,
To enable disease monitoring in cancer patients using LB, we established the LOQ for the LB Duplex sequencing workflow. The LOQ represents the cutoff above which VAFs can be accurately quantified based on acceptable trueness (>90%) and precision (>80%) (
To estimate trueness, representing the closeness of agreement between measured and reference VAF, we calculated the bias between actual VAFs (confirmed by digital droplet PCR) and measured VAFs of variants present in each plasma reference material. Variants with ~0.5% VAF were determined with 85.0% trueness, variants with ~1% VAF with trueness of 92.5%, and variants with ~5% VAF with 99.9% trueness (
We further established precision in terms of repeatability based on each variant present in reference materials. Therefore, we calculated the pooled standard deviation over all variants between the two replicates of each reference material. For variants with ~0.5% VAF, ~1% and ~5% VAF repeatability was determined to be 34.4%, 65.2%, and 91.9%, respectively (
Based on these results we were able to establish the LOQ as cutoff for VAF quantification at a VAF of 5% with a total error of 16.2%. These results indicate that variants determined with VAFs ≥5% can be reliably quantified with our LB Duplex sequencing workflow and therefore are informative for disease monitoring. Since tissue analysis cannot be used for disease monitoring, we only validated the tissue analysis with qualitative rather than quantitative intent. However, measured VAFs in two replicates for most of the variants deviated only 0.2% to 7.9%. Only for the
To test the diagnostic value of our approach, we applied plasma and tissue analysis with our combined workflow in samples of a ten-year old girl with clinically diagnosed asymmetric overgrowth syndrome including arteriovenous malformations in the right arm and right leg, but without molecular diagnosis that would support personalized treatment (
Clinically confirmed overgrowth syndrome in a 10-year-old girl to be analyzed for molecular clarification. Marked asymmetry of hands and legs. The feet have a difference in circumference. On the back, there is a large asymmetric reddish-brown area of skin, with temperature-depended hyperpigmentation, suggestive of capillary malformations in the area.
Initially, standard molecular diagnostics were performed including karyotyping from heparin blood, which provided inconspicuous results and microarray analysis from EDTA blood that identified a duplication of 15q13.3 not related to the phenotype. The following WES from skin fibroblasts did not identify any disease-causing variant. Using our tissue analysis workflow including 17 genes associated with overgrowth syndrome, also no pathogenic variant could be identified, whereas LB analysis identified
Molecular analysis of clinically confirmed overgrowth syndrome.
Method | Result | Variant | Measured VAF | Material | LOD |
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unsuspicious | – | – | Heparin blood | |
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VUS (CNV - not related to phenotype) | Duplication of 15q13.3 | – | EDTA blood | |
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unsuspicious | – | – | Skin fibroblasts | 20% |
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unsuspicious | – | – | Skin fibroblasts | 8% |
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SNV pathogenic |
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Plasma | 0.5% |
Analyzed genes: aAKT1, AKT3, GNAQ, GNAS, IDH1, IDH2, IKBKG, KRAS, MTOR, NF1, NRAS, NSDHL, PIK3CA, PIK3R2, PORCN, PTCH1. PTEN, RASA1, SPRED1, TSC1, TSC2; bHRAS, FGFR1, KRAS, AKT3, BRAF, MTOR, CCND2, GNA11, GNAQ, NRAS, MAP2K1, RASA1, EPHB4, PIK3R2, SMO, PIK3CA, GNAS;
LB is a promising tool in precision medicine. It is fast, non-invasive and represents disease heterogeneity at any desired time point (
A major challenge for implementation of highly sensitive LB analysis into clinical practice are the generally high costs due to the high sequencing coverage required. In our workflow, one to two LB samples can be pooled with seven tissue samples on an Illumina NextSeq flowcell (Mid output), at reasonable cost. Notably, combining 17 plasma samples on a NovaSeq SP flowcell (i.e. the maximal number of LB samples that can be pooled on this flowcell) reduces costs per sample significantly, which is in the range of targeted LB hotspot analysis using digital droplet PCR and tissue sample analysis. Notably, adding the option of LB analysis to clinical labs not only results in higher flexibility but also reduces costs for tissue sample analyses. Taken together, to the best of our knowledge this is the most cost-effective approach for a mid-size and highly sensitive NGS LB panel.
Using our workflow, LB samples can be processed in parallel to tissue samples similar to the MSI-ACCESS assay developed by the Memorial Sloan Kettering Cancer Center (MSK), processing LB samples from cancer patients in parallel to white blood cells (WBC) (
Rather than only including actionable variants for mosaic disease, we show with our case report a clinical proof-of-concept that LB can be extended from cancers to all heterogeneous diseases such as mosaic diseases, including asymmetric overgrowth syndromes (e.g. Proteus syndrome, Klippel-Trennaunay syndrome and
However, there are limitations that need to be considered. Accurate detection of variants <0.5% VAF is challenging. This finding is in line with a recent study testing five leading commercial ctDNA assays, which show generally high performance for VAFs ≥0.5% (
In conclusion, LB is capable to detect the complete mutational profile of both the primary tumor and metastatic lesions. LB Duplex sequencing pushes the boundaries for detection of low frequency variants in plasma with NGS based analysis. Our broad Duplex sequencing panel enables highly sensitive detection of therapy relevant variants in tumor and mosaic diseases. We were able to identify the
The sequence data have been deposited at the European Genome-phenome Archive (EGA) under accession number EGAS00001006805. The original contributions presented in the study are included in the article/
The studies involving human participants were reviewed and approved by ethics commission of the Bavarian Medical Association. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.
TN, VS-L, and JP researched the literature to identify cancer and PROS associated genes. FS established the target region of the sequencing panel. AH developed the experimental procedures, and performed all experiments. AH, AL, RE, BL, and JP analyzed and interpreted the data. AH, RE, and BL performed statistical analysis. AH and JP designed the study. JP supervised the work. EH-F provided financial and technical resources to enable conduction of the study. AH and JP wrote the manuscript. All authors contributed to the article and approved the submitted version.
We would like to thank the individuals providing their informed consent to use their DNA samples for research purposes.
Authors RE and BL were employed by Limbus Medical Technologies GmbH.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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