An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants

Stasik, Sebastian; Schuster, Caroline; Ortlepp, C.; Platzbecker, Uwe; Bornhäuser, Martin; Schetelig, Johannes; Ehninger, Gerhard; Folprecht, Gunnar; Thiede, Christian

doi:10.1016/j.bdq.2017.12.001

Cited by 39 publications

(30 citation statements)

References 42 publications

(49 reference statements)

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“…This can decrease the limit of detection, as true mutations can be distinguished from PCR errors or sequencing errors. When using optimal technical conditions for NGS, including unique molecular identifiers, detection rates of 0.01–0.0015% have been documented for both IonTorrent NGS and ddPCR . Finally, it seems sensible to combine ctDNA and other biomarkers in one test, attempting to increase sensitivity and specificity.…”

Section: Discussionmentioning

confidence: 99%

“…When using optimal technical conditions for NGS, including unique molecular identifiers, detection rates of 0.01-0.0015% have been documented for both IonTorrent NGS and ddPCR. 46,47 Finally, it seems sensible to combine ctDNA and other biomarkers in one test, attempting to increase sensitivity and specificity. An interesting illustration of this approach in the diagnostic setting is the CancerSEEK test, which used a combination of ctDNA and established serum makers to detect early stage cancers, showing a sensitivity of 69-98% for the detection of five cancer types, including pancreatic cancer.…”

Section: Discussionmentioning

confidence: 99%

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Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

Strijker

Soer

Pastena

et al. 2019

Intl Journal of Cancer

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Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow‐up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell‐free DNA from plasma samples of 58 treatment‐naive mPDAC patients was isolated and sequenced using a custom‐made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross‐check with droplet digital PCR showed good agreement in Bland–Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6–4.9) versus 8.4 (95% CI 1.6–15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01–1.09, p = 0.005; HR 1.00, 95% CI 1.01–1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

Strijker

Soer

Pastena

et al. 2019

Intl Journal of Cancer

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“…For genomic and transcriptomic characterization of CTCs, the purity of the CTCs-enriched fraction is the most important factor, since background signal might interfere with the readout of the molecular assay. For genomic test, the sensitivity of the assay for mutation detection in the background of wild-type allele should be considered [75,76]; though, in practice, genomic testing of CTCs is usually performed at the single cell level. In the transcriptomic assays, expression of genes included in the molecular test should be analysed in a number of control samples to adjust for background expression level and apply a cut-off value, when appropriate [62,63,64,77].…”

Section: Methods Of Ctcs Isolationmentioning

confidence: 99%

Profiling of Invasive Breast Carcinoma Circulating Tumour Cells—Are We Ready for the ‘Liquid’ Revolution?

Braun

Markiewicz

Kordek

et al. 2019

Cancers

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As dissemination through blood and lymph is the critical step of the metastatic cascade, circulating tumour cells (CTCs) have attracted wide attention as a potential surrogate marker to monitor progression into metastatic disease and response to therapy. In patients with invasive breast carcinoma (IBC), CTCs are being considered nowadays as a valid counterpart for the assessment of known prognostic and predictive factors. Molecular characterization of CTCs using protein detection, genomic and transcriptomic panels allows to depict IBC biology. Such molecular profiling of circulating cells with increased metastatic abilities appears to be essential, especially after tumour resection, as well as in advanced disseminated disease, when information crucial for identification of therapeutic targets becomes unobtainable from the primary site. If CTCs are truly representative of primary tumours and metastases, characterization of the molecular profile of this easily accessible ‘biopsy’ might be of prime importance for clinical practice in IBC patients. This review summarizes available data on feasibility and documented benefits of monitoring of essential IBC biological features in CTCs, with special reference to multifactorial proteomic, genomic, and transcriptomic panels of known prognostic or predictive value.

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“…The PCR reactions were purified using a tworound purification process with Agencourt AMPure XP Reagent (Beckman Coulter) and eluted in 30 to 50 mL ddH 2 O. The barcoded PCR products were quantified with a Qubit 2.0 fluorometer (Life Technologies) using the Qubit dsDNA HS Assay (Life Technologies) and sequenced unidirectionally on an Ion Torrent PGM NGS system (Life Technologies), according to the manufacturer's protocols (16).…”

Section: Library Preparation Sequencing and Data Analysismentioning

confidence: 99%

TERT Promoter Mutation Detection in Cell-Free Tumor-Derived DNA in Patients with IDH Wild-Type Glioblastomas: A Pilot Prospective Study

Juratli

Stasik

Zolal

et al. 2018

Clinical Cancer Research

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We conducted a pilot study to assess the feasibility and the potential implications of detecting promoter (p)-mutant cell-free tumor-derived DNA (tDNA) in the cerebrospinal fluid (CSF) and plasma of glioblastoma patients. Matched CSF and plasma samples were collected in 60 patients with glial tumors. The CSF collection was obtained during surgery, before any surgical manipulation of the tumor. The extracted tDNA and corresponding tumor DNA samples were analyzed for p and isocitrate dehydrogenase ( hotspot mutations. In addition, the variant allele frequency (VAF) of p mutation in the CSF-tDNA was correlated with tumor features and patients' outcome. Thirty-eight patients had p-mutant/ wild-type glioblastomas. The matched p mutation in the CSF-tDNA was successfully detected with 100% specificity (95% CI, 87.6-100%) and 92.1% sensitivity (95% CI, 78.6-98.3%) ( = 35/38). In contrast, the sensitivity in the plasma-tDNA was far lower [ = 3/38, 7.9% (95% CI, 1.6-21.4%)]. We concordantly observed a longer overall survival of patients with low VAF in the CSF-tDNA when compared with patients with high VAF, irrespective of using the lower quartile VAF [11.45%; 14.0 mo. (95% confidence interval, CI, 10.3-17.6) vs. 8.6 mo. (95% CI, 4.1-13.2), = 0.035], the lower third VAF [13%; 15.4 mo. (95% CI, 11.6-19.2) vs. 8.3 mo. (95% CI, 2.3-14.4), = 0.008], or the median VAF [20.3%; 14.0 mo. (95% CI, 9.2-18.7) vs. 8.6 mo. (95% CI, 7.5-9.8), = 0.062] to dichotomize the patients. This pilot study highlights the value of CSF-tDNA for an accurate and reliable detection of p mutations. Furthermore, our findings suggest that highp mutation VAF levels in the CSF-tDNA may represent a suitable predictor of poor survival in glioblastoma patients. Further studies are needed to complement the findings of our exploratory analysis. .

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An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants

Cited by 39 publications

References 42 publications

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

Profiling of Invasive Breast Carcinoma Circulating Tumour Cells—Are We Ready for the ‘Liquid’ Revolution?

TERT Promoter Mutation Detection in Cell-Free Tumor-Derived DNA in Patients with IDH Wild-Type Glioblastomas: A Pilot Prospective Study

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