scholarly journals Rapid identification of genomic structural variations with nanopore sequencing enables blood-based cancer monitoring

2019 ◽  
Author(s):  
Jose Espejo Valle-Inclan ◽  
Christina Stangl ◽  
Anouk C. de Jong ◽  
Lisanne F. van Dessel ◽  
Markus J. van Roosmalen ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Rapid Development ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Rapid Identification ◽  
Cancer Type ◽  
Nanopore Sequencing ◽  
Structural Variations ◽  
Liquid Biopsies ◽  
Random Forest Classification

AbstractSomatic genomic structural variations (SVs) are promising personalized biomarkers for sensitive and specific detection of circulating tumor DNA (ctDNA) in liquid biopsies. However, affordable and fast identification of such SV biomarkers is challenging, which hinders routine use in the clinic. Here, we developed a novel approach - termed SHARC - for rapid discovery of somatic SVs as personalized tumor biomarkers. SHARC combines low coverage cancer genome sketching by using Oxford Nanopore sequencing with random forest classification and a dedicated filtering pipeline to enrich for somatic SVs. Our method leverages the real-time and long-read capabilities of nanopore sequencing to identify somatic SV breakpoints at nucleotide resolution from a tumor tissue biopsy within three days. We applied SHARC to tumor samples of high-grade ovarian and prostate cancer and validated on average ten somatic SVs per sample with PCR mini-amplicons. An accompanying method for SV breakpoint detection from liquid biopsies was devised based on digital PCR, enabling detection of cancer in a quantitative manner. Using this method, we retrospectively monitored treatment response in patients with metastatic prostate cancer. Our work demonstrates that SHARC forms a universal framework for rapid development of personalized biomarker assays for blood-based monitoring of any cancer type.

Genomic immunotherapy (IO) biomarkers detected on comprehensive genomic profiling (CGP) of tissue and circulating tumor DNA (ctDNA).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2541-2541
Author(s):  
Takayuki Yoshino ◽  
Hanna Tukachinsky ◽  
Jessica Kim Lee ◽  
Ethan Sokol ◽  
Dean C. Pavlick ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Predictive Biomarkers ◽  
Principal Component ◽  
Pathogenic Mutation ◽  
Circulating Tumor Dna ◽  
Driver Mutations ◽  
Cancer Type ◽  
Liquid Biopsies ◽  
Mutational Burden ◽  
Relative Prevalence

2541 Background: The dramatic impact of IO on treatment outcomes has heightened interest in predictive biomarkers, including genomic markers such as tumor mutational burden (TMB) and microsatellite instability (MSI). The recent FDA approval of pembrolizumab for previously treated advanced solid tumors with elevated TMB (≥10 mut/Mb on FoundationOne CDx, F1CDx) now requires a better understanding of the prevalence of this and other IO biomarkers detected on CGP, including differences between TMB detected in tissue and mutational burden detected in blood (bTMB). Methods: Tissue and plasma biopsies were profiled with two CGP panels of 324 genes with 0.8 Mb genome coverage (F1CDx and FoundationOne LiquidCDx). Mutational burden was calculated by counting somatic variants (single nucleotide and indels, including synonymous variants, excluding germline and driver mutations) with variant allele frequency (VAF) ≥5% in tissue (TMB) or ≥0.5% in ctDNA (bTMB). MSI score was assessed using 95 repetitive loci and principal component analysis (tissue) or >1,800 repetitive loci (plasma). ctDNA levels were estimated using composite tumor fraction (cTF), a metric based on aneuploidy and VAF. Results: Pan-cancer, TMB ≥10 was detected in 19% of tissue cases (29,238/156,294) and was common in melanoma (53%), small cell (41%), NSCLC (40%), bladder (39%), and endometrial (24%). bTMB ≥10 was detected in 13% of liquid biopsies (806/6,295); prevalence by cancer type was correlated with prevalence of elevated TMB (r = 0.81). Samples with bTMB ≥10 had an elevated cTF (median 13%, IQR 5 - 31%) as compared to samples with bTMB <10 (median 1.8%, IQR 0.6 - 7%, p < 0.001). Among 353 cases with both tissue and liquid CGP results (median 11 months apart), the relative prevalence of TMB ≥10 (12%) and bTMB ≥10 (13%) were similar, with concordant detection in 303 cases (86%). MSI-high (MSI-H) was seen in 2.2% of tissue CGP (3,461/156,294), most often in endometrial (19%), stomach (6.0%), and colorectal (5.3%) cancers, while MSI-H was detected in 0.68% of ctDNA specimens (43/6,295), which were also those with elevated cTF (median 11%, IQR 7 - 23%). Of 3,504 cases with MSI-H signature on tissue or liquid CGP, 1,619 (46%) had a pathogenic mutation detected in MLH1/MSH2/MSH6/PMS2 (15% predicted germline). CD274 amplification was detected in 1,207 cases (0.77%) of tissue CGP and 11 cases (0.17%) in ctDNA. Conclusions: Elevated bTMB is overall less prevalent than elevated tissue TMB, though these biomarkers are detected in similar cancer types. Detection of bTMB ≥10 and MSI-H in liquid biopsy was associated with elevated ctDNA levels, suggesting a limit of detection, and potentially indicating a more aggressive biology in samples positive for these biomarkers. Further investigation is needed to understand the utility of bTMB for identifying high TMB tumors that may benefit from IO.

scholarly journals High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies

2020 ◽  
Vol 66 (4) ◽  
pp. 606-613 ◽  
Author(s):  
Amanda Bortolini Silveira ◽  
François-Clément Bidard ◽  
Amélie Kasperek ◽  
Samia Melaabi ◽  
Marie-Laure Tanguy ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Large Scale ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Diagnostic Tools ◽  
Tumor Biomarker ◽  
Analytical Sensitivity ◽  
Liquid Biopsies ◽  
Tumor Dna ◽  
Large Scale Screening

Abstract Background Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. Methods We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. Results The 3 ddPCR assays reached analytical sensitivity &lt;0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. Conclusions This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

scholarly journals Detection of AR-V7 in Liquid Biopsies of Castrate Resistant Prostate Cancer Patients: A Comparison of AR-V7 Analysis in Circulating Tumor Cells, Circulating Tumor RNA and Exosomes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 688 ◽  
Author(s):  
Mohammed Nimir ◽  
Yafeng Ma ◽  
Sarah A. Jeffreys ◽  
Thomas Opperman ◽  
Francis Young ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Digital Pcr ◽  
Liquid Biopsies ◽  
Tumor Biopsy ◽  
Highly Sensitive ◽  
Castrate Resistant Prostate Cancer ◽  
Castrate Resistant

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker.

scholarly journals Plasma Circulating Tumor DNA Levels for the Monitoring of Melanoma Patients: Landscape of Available Technologies and Clinical Applications

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Benoit Busser ◽  
Julien Lupo ◽  
Lucie Sancey ◽  
Stéphane Mouret ◽  
Patrice Faure ◽  
...  
Keyword(s):  
Acquired Resistance ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Clinical Applications ◽  
Molecular Heterogeneity ◽  
Liquid Biopsies ◽  
Melanoma Patients ◽  
Ctdna Analysis ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations inBRAF,NRAS, orKITare present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

scholarly journals Epigenetic Analysis of Circulating Tumor DNA in Localized and Metastatic Prostate Cancer: Evaluation of Clinical Biomarker Potential

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1362
Author(s):  
Marianne Trier Bjerre ◽  
Maibritt Nørgaard ◽  
Ole Halfdan Larsen ◽  
Sarah Østrup Jensen ◽  
Siri H. Strand ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
De Novo ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Healthy Controls ◽  
Tissue Samples ◽  
Castration Resistant ◽  
Clinical Biomarker ◽  
Epigenetic Analysis ◽  
Tumor Dna

Novel and minimally-invasive prostate cancer (PCa)-specific biomarkers are needed to improve diagnosis and risk stratification. Here, we investigated the biomarker potential in localized and de novo metastatic PCa (mPCa) of methylated circulating tumor DNA (ctDNA) in plasma. Using the Marmal-aid database and in-house datasets, we identified three top candidates specifically hypermethylated in PCa tissue: DOCK2, HAPLN3, and FBXO30 (specificity/sensitivity: 80%–100%/75–94%). These candidates were further analyzed in plasma samples from 36 healthy controls, 61 benign prostatic hyperplasia (BPH), 102 localized PCa, and 65 de novo mPCa patients using methylation-specific droplet digital PCR. Methylated ctDNA for DOCK2/HAPLN3/FBXO30 was generally not detected in healthy controls, BPH patients, nor in patients with localized PCa despite a positive signal in 98%–100% of matched radical prostatectomy tissue samples. However, ctDNA methylation of DOCK2, HAPLN3, and/or FBXO30 was detected in 61.5% (40/65) of de novo mPCa patients and markedly increased in high- compared to low-volume mPCa (89.3% (25/28) vs. 32.1% (10/31), p < 0.001). Moreover, detection of methylated ctDNA was associated with significantly shorter time to progression to metastatic castration resistant PCa, independent of tumor-volume. These results indicate that methylated ctDNA (DOCK2/HAPLN3/FBXO30) may be potentially useful for identification of hormone-naïve mPCa patients who could benefit from intensified treatment.

scholarly journals Detection of Microsatellite Instability via Circulating Tumor DNA and Response to Immunotherapy in Metastatic Castration-Resistant Prostate Cancer: A Case Series

2021 ◽  
pp. 190-196
Author(s):  
Deepak Ravindranathan ◽  
Greta Anne Russler ◽  
Lauren Yantorni ◽  
Leylah M. Drusbosky ◽  
Mehmet Asim Bilen
Keyword(s):  
Prostate Cancer ◽  
Microsatellite Instability ◽  
Case Series ◽  
Circulating Tumor Dna ◽  
Castration Resistant Prostate Cancer ◽  
Liquid Biopsies ◽  
Castration Resistant ◽  
Before And After ◽  
Initiation Of Therapy ◽  
Tumor Dna

Pembrolizumab has been approved by the US Food and Drug Administration for the treatment of metastatic or unresectable solid tumors that are microsatellite instability-high (MSI-H) or mismatch repair deficient. Blood-based circulating tumor DNA (ctDNA) assays have been validated to identify tumors with MSI-H status without the need for tissue biopsy. We report 2 patients with metastatic castration-resistant prostate cancer (mCRPC) who had prior treatment with multiple lines of therapy and underwent ctDNA testing, which detected MSI-H status. Both patients were treated with pembrolizumab, resulting in an excellent clinical response measured with liquid biopsies before and after initiation of therapy, which demonstrated a significant reduction in somatic-variant allele frequency in addition to a decrease in prostate serum antigen levels.

Greater than two coexisting mutations in KRAS and NRAS identified in the circulating tumor DNA fraction of liquid biopsies by NGS and confirmed with ddPCR.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15563-e15563
Author(s):  
Hala Boulos ◽  
Robert Tell ◽  
Nike Beaubier ◽  
Richard Blidner
Keyword(s):  
Liquid Biopsy ◽  
Detection Threshold ◽  
Treatment Resistance ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Precision Oncology ◽  
Single Nucleotide Variants ◽  
Liquid Biopsies ◽  
Low Frequencies ◽  
Tumor Dna

e15563 Background: Liquid biopsies are increasingly utilized as a non-invasive tool in precision oncology to assess tumor mutational profiles in order to select targeted therapies, detect treatment resistance, and monitor disease progression in cancer patients. Additionally, liquid biopsies may provide a more comprehensive representation of tumor heterogeneity than standard tissue biopsies. However, limitations such as scarcity of circulating tumor DNA (ctDNA) and/or variants at low frequencies can be technically challenging to detect by next-generation sequencing (NGS) assays. Here, we use NGS to detect greater than two KRAS/NRAS mutations coexisting in single samples at low variant allele frequencies (VAFs). Methods: The Tempus xF liquid biopsy NGS assay is designed to detect actionable oncologic targets spanning 105 genes in plasma. The assay was validated to reliably detect single-nucleotide variants at 0.25% VAF, indels and copy number variants at 0.5% VAF, and fusions at 1% VAF with 96.2%-100% specificity and 97.4%-100% sensitivity. Pre-designed digital PCR assays were modified to measure 10ng of cell-free DNA (cfDNA) on a droplet-digital PCR (ddPCR) platform. Results: Overall, we report 100% positive predictive value and high correlation between ddPCR results and xF VAF, as well as in individual variants, such as KRAS G12D. Unexpectedly, we detected more than two coexisting KRAS/NRAS mutations at a low VAF in the plasma samples. To orthogonally confirm these results, ddPCR was deployed to independently measure the presence of each cfDNA variant with a sensitivity of 0.09% VAF. Subsequent ddPCR analysis of all targeted variants were concordant with NGS results. Conclusions: The occurrence of multiple KRAS and NRAS mutations in a single sample is quite uncommon and may be falsely interpreted as an NGS artifact. However, verification of this phenomenon by ddPCR confirmed the validity of the NGS liquid biopsy approach. These results highlight the capability of the Tempus xF assay to detect low-frequency variants, including those that fall below the validated detection threshold, which is essential for the diagnosis of early disease.

scholarly journals The detection of specific hypermethylated WIF1 and NPY genes in circulating DNA by crystal digital PCR™ is a powerful new tool for colorectal cancer diagnosis and screening

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexis Overs ◽  
Mylène Flammang ◽  
Eric Hervouet ◽  
Laurent Bermont ◽  
Jean-Luc Pretet ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Point Of View ◽  
University Hospital ◽  
Liquid Biopsies ◽  
Tissue Samples ◽  
Mutation Status ◽  
Tumor Tissues ◽  
Tumor Stages

Abstract Background In oncology, liquid biopsy is of major relevance from theranostic point of view. The searching for mutations in circulating tumor DNA (ctDNA) in case of colorectal cancers (CRCs) allows the optimization of patient care. In this context, independent of mutation status biomarkers are required for its detection to confirm the presence of ctDNA in liquid biopsies. Indeed, the hypermethylation of NPY and WIF1 genes appear to be an ideal biomarker for the specific detection of ctDNA in CRCs. The objective of this work is to develop the research of hypermethylation of NPY and WIF1 by Crystal Digital PCR™ for the detection of ctDNA in CRCs. Methods Detection of hypermethylated NPY and WIF1 was developed on Cristal digital PCR™. Biological validation was performed from a local cohort of 22 liquid biopsies and 23 tissue samples from patients with CRC. These patients were treated at the University Hospital of Besancon (France). Results The local cohort study confirmed that NPY and WIF1 were significantly hypermethylated in tumor tissues compared to adjacent non-tumor tissues (WIF1 p < 0.001; NPY p < 0.001; non-parametric Wilcoxon paired-series test). Histological characteristics, tumor stages or mutation status were not correlated to the methylation profiles. On the other hand, hypermethylation of NPY or WIF1 in liquid biopsy had a 95.5% [95%CI 77–100%] sensitivity and 100% [95%CI 69–100%] specificity. Conclusion Using Crystal digital PCR™, this study shows that hypermethylation of NPY and WIF1 are constant specific biomarkers of CRCs regardless of a potential role in carcinogenesis.

scholarly journals Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR

2022 ◽  
Vol 11 (1) ◽  
pp. 257
Author(s):  
Francis P. Young ◽  
Therese M. Becker ◽  
Mohammed Nimir ◽  
Thomas Opperman ◽  
Wei Chua ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Splice Variants ◽  
Point Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Castration Resistant Prostate Cancer ◽  
Liquid Biopsies ◽  
Castration Resistant ◽  
Hormone Sensitive

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

scholarly journals Optimizing Nanopore sequencing-based detection of structural variants enables individualized circulating tumor DNA-based disease monitoring in cancer patients

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Jose Espejo Valle-Inclan ◽  
Christina Stangl ◽  
Anouk C. de Jong ◽  
Lisanne F. van Dessel ◽  
Markus J. van Roosmalen ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Structural Variants ◽  
Cancer Genome Sequencing ◽  
Novel Approach ◽  
Oxford Nanopore ◽  
Response Biomarkers ◽  
Low Coverage

AbstractHere, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay. The results suggest that SV dynamics correlate with and may improve existing treatment-response biomarkers such as PSA.https://github.com/UMCUGenetics/SHARC.

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