The authors would like to acknowledge the generosity of all patients and their families. This work was supported by funding from the Smashing Walnuts Foundation (Middleburg, VA), the V Foundation (Atlanta, GA), The Gabriella Miller Kids First Data Resource Center, Clinical and Translational Science Institute at Children&#39;s National (5UL1TR001876-03), Musella Foundation (Hewlett, NY), Matthew Larson Foundation (Franklin Lake, NJ), The Lilabean Foundation for Pediatric Brain Cancer Research (Silver Spring, MD), the Childhood Brain Tumor Foundation (Germantown, MD), the Children&#39;s Brain Tumor Tissue Consortium (Philadelphia, PA), and Rally Foundation for Childhood Cancer Research (Atlanta, GA).

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PNOC003: Precision medicine trial for children with diffuse intrinsic pontine glioma.</a> Neuro-Oncology. 19 (4), iv14 (2017).</li><li>Kilburn, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIPG-76.+PNOC-003:+Precision+medicine+trial+for+children+with+diffuse+intrinsic+pontine+glioma:+preliminary+experience+with+multi-agent+personalized+therapy+recommendations.">DIPG-76. PNOC-003: Precision medicine trial for children with diffuse intrinsic pontine glioma: preliminary experience with multi-agent personalized therapy recommendations.</a> Neuro-Oncology. 20 (2), i64 (2018).</li><li>Aquino, D., Gioppo, A., Finocchiaro, G., Bruzzone, M. 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A., Wengel, J., Nielsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LNA+(locked+nucleic+acids):+synthesis+and+high-affinity+nucleic+acid+recognition.">LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition.</a> Chemical Communications. 29 (4), 455-456 (1998).</li><li>Owczarzy, R., You, Y., Groth, C. L., Tataurov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+and+mismatch+discrimination+of+locked+nucleic+acid-DNA+duplexes.">Stability and mismatch discrimination of locked nucleic acid-DNA duplexes.</a> Biochemistry. 50 (43), 9352-9367 (2011).</li><li>Priya, N. 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T., Pfundheller, H. M., Choleva, Y., Kongsbak, L., Moller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+locked+nucleic+acid+(LNA).">Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).</a> Expert Review of Molecular Diagnostics. 3 (1), 27-38 (2013).</li><li>Ugozzoli, L. A., Latorra, D., Puckett, R., Arar, K., Hamby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+genotyping+with+oligonucleotide+probes+containing+locked+nucleic+acids.">Real-time genotyping with oligonucleotide probes containing locked nucleic acids.</a> Analytical Biochemistry. 324 (1), 143-152 (2004).</li><li>Pan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+tumors+of+the+brainstem+by+sequencing+of+CSF-derived+circulating+tumor+DNA.">Molecular profiling of tumors of the brainstem by sequencing of CSF-derived circulating tumor DNA.</a> Acta Neuropathologica. , (2018).</li><li>Stewart, C. M., Tsui, D. W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+DNA+for+non-invasive+cancer+management.">Circulating cell-free DNA for non-invasive cancer management.</a> Cancer Genetics. , (2018).</li><li>Barault, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+methylated+circulating+DNA+biomarkers+for+comprehensive+non-invasive+monitoring+of+treatment+response+in+metastatic+colorectal+cancer.">Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer.</a> Gut. 67 (11), 1995-2005 (2018).</li></ol>

The method described in this manuscript is included in an International patent application "Methods for detecting cancer biomarkers" by Javad Nazarian &amp; Eshini Panditharatna.

Here we have presented our method for detecting and quantifying the allelic frequency of tumor mutations in cfDNA from&#160;patient liquid biopsy. We emphasize critical steps for the success of this method, including pre-analytical sample processing, cfDNA extraction, PCR assay design, and data analysis. To limit the sample volume used, cfDNA is extracted from 1 mL of plasma but only 500 &#181;L of CSF. When extracting from CSF, the protocol for extraction from 1 mL of urine (following the cfDNA extraction kit handbook12) is used, as per the manufacturer&#39;s recommendation. The difference in sample volume required between plasma and CSF is due to lower levels of tumor-specific cfDNA in plasma compared to CSF of patients with brain tumors, necessitating larger sample volumes for mutation detection8. If the sample is available, more than 1 mL of plasma may be extracted from to produce higher DNA yield. However, in the case of pediatric patients, it is important to minimize the amount of blood used whenever possible, as even a simple procedure such as blood draw is fatiguing to pediatric patients with cancers undergoing radiation therapies. Extracting from 1 mL aliquots of plasma also enables replicate extractions, such that the assay can be repeated (e.g., in cases of failure in DNA extraction or sample contamination).
In an effort to reduce any sample use that is not strictly necessary, cfDNA is typically not quantified. However, we have found a range of cfDNA concentrations between 0.2-2 ng/&#181;L and 0.6-13 ng/&#181;L in plasma and CSF, respectively. Given the low amount of cfDNA, and the fact that tumor-specific mutant alleles are present at a low frequency, a pre-amplification step using the same set of primers used for dPCR is necessary to significantly increase the amount of target DNA in the sample, aiding in mutation detection8. Diluting the pre-amplified product in DNA suspension buffer provides sufficient volume for technical replicates, which aids in distinguishing true positives. Because the number of mutant droplets can be low (for example, between 0-2 mutant droplets in a plasma sample), the inclusion of technical replicates is key for resolving mutation status. While one PCR well may yield 0 mutant droplets, the other two may yield 1-2 for a single plasma sample analyzed in triplicate; thus, the inclusion of replicates allows for greater accuracy when determining mutation status. The MAF is then calculated as the average of the replicate values.
Multiplexed pre-amplification (preamplifying wildtype and mutant alleles of two mutations of interest) increases the utility of a single sample, as the same starting material can be used to test for two mutations. Importantly, a multiplex preamplification product can be analyzed in singleplex during dPCR for greater simplicity, as described here. However, both preamplification PCR and dPCR may be multiplexed. When multiplexing, conditions must be optimized for both sets of primers and probes: primer annealing temperatures must be similar to run together in PCR amplification, and probes should be designed to generate distinct clusters (based on fluorescent signal and intensity). When validating a new set of primers and probes, run an annealing temperature gradient to determine optimum annealing temperature based on the range suggested by the manufacturer. Before testing probes on patient cfDNA specimens, validate them using synthetic DNA constructs and/or tumor tissue gDNA of different inputs (i.e., up to 10 ng).
For the detection of mutant alleles with greater specificity and reduced mismatches in the hybridization of probe to the target DNA, locked nucleic acid (LNA) probes are used. LNA is a nucleic acid analog with a methylene bridge connecting the 2&#39;-oxygen and 4&#39;-carbon of the ribose ring9. The methylene bridge locks the nucleic acid into a rigid bicyclic conformation that restricts flexibility, increases thermal duplex stability, and improves the specificity of probe hybridization to target DNA10,18,19. If a single base mismatch exists between LNA probe and template DNA, duplex formation between probe and target will destabilize. As such, LNA probes improve the specificity of probe binding and result in a higher signal-to-noise ratio11. Analysis of plasma and CSF from non-CNS-diseased pediatric patients has established the specificity of our assay with LNA probes targeting H3F3A p.K27M, to determine that an allelic frequency of equal to or less than 0.001% is considered a false positive8. For additional considerations for optimizing the design of probes and primers, including GC content, amplicon size, probe reporter dyes, and quenchers, refer to the dPCR manufacturer guidelines14,17. Although our method is optimized for use with the RainDance system, the protocol may be adapted for use with other dPCR platforms.
The method presented here draws strength from the high sensitivity and target enrichment achieved by dPCR, which remains the platform of choice for detecting rare tumor mutations in cfDNA. Although powerful, dPCR is limited in the number of mutations that can be tested for in a single assay. An alternative to dPCR is next generation sequencing (NGS), which may detect multiple mutations across many genes, increasing the utility of a single sample. NGS can detect mutations in CSF and plasma of patients with brainstem gliomas20, however, is currently less sensitive than dPCR at detecting tumor mutations in cfDNA, with detection limits of 0.1-10% MAF compared to 0.001% in PCR-based approaches21. dPCR also achieves faster turnaround time than NGS, enabling rapid detection of mutations of interest.The PCR approach is applicable across cancer types and can be expanded to detect hotspot mutations and methylated cfDNA22.
The liquid biopsy is indeed in its infancy and will require further development for tailoring to specific diseases. Tumor monitoring in the context of tumor evolution will be the next challenge, where a platform capable of detecting emerging mutations with high sensitivity would be required. Additionally, platforms capable of detecting a variety of biomolecules (peptides, cytokines, RNA) will be highly beneficial in assessing tumor response to treatment, as well as advancing personalized clinical interventions.

Due to&#160;limitations in the availability of&#160;tumor tissue for molecular analyses, there is a need for the development of highly sensitive methods capable of detecting tumor somatic mutations in patient biofluids, including plasma, serum and cerebrospinal fluid (CSF). For example, pediatric diffuse midline gliomas (DMGs) present a challenge due to their neuroanatomical location. Over the past decade, molecular profiling of DMG tissue specimens has uncovered driver mutations in this tumor type1 and has revealed spatial and temporal heterogeneity of mutations, making biopsy necessary for characterizing a patient within a disease subgroup and promoting molecularly-targeted therapies2. As such, clinical trials advocate for integrating surgical biopsies for tumor molecular profiling at upfront diagnosis3,4,5,6. During&#160;treatment, monitoring of&#160;therapeutic response in DMGs is limited to MRI, which lacks sensitivity to detect small changes or tumor genomic evolution. MRI is also prone to detect pseudoprogression, transient inflammation at the site of the tumor that mimics true progression on imaging and may misinform interpretation of tumor response7. Thus, DMGs represent a tumor type with a high need for an alternative, minimally invasive means of detecting tumor mutations and monitoring clinical response. In order to address these needs, we developed and optimized a protocol for detecting, and quantifying the allelic frequency of, tumor mutations in cfDNA from plasma, serum and CSF of children with midline gliomas8. Given that childhood DMGs often (&#62;80%) harbor a somatic lysine-to-methionine mutation at position 27 of histone variant H3.1 (H3.1K27M, 20% of cases) or H3.3 (H3.3K27M, 60% of cases)1, these and other mutations characteristic of DMGs (i.e., ACVR1, PIK3R1) were targeted for mutant allele quantification using cfDNA8. This assay can be tailored to detect hotspot mutations in other tumor types using sequence-specific primers and probes. The versatility of this approach makes it applicable to a variety of cancers which would benefit from the integration of cfDNA-based diagnostic tools and therapeutic monitoring.
To sensitively detect low allelic frequency mutations in cfDNA, we employ a droplet digital PCR (dPCR)-based approach. In this method, the PCR reaction mixture is partitioned into a theoretical maximum of 10 million droplets using the dPCR platform (e.g., RainDance), with 7-9 million droplets per PCR well typically seen experimentally. Due to the high degree of&#160;sample partitioning, at most only one to two DNA molecules can be present in each droplet. PCR amplification and sequence-specific fluorescent probe hybridization can occur in each droplet, such that millions of reactions take place. This maximizes the sensitivity and enables detection of rare mutant alleles, which might otherwise go undetected against a background of wildtype DNA. The utilization of locked nucleic acid (LNA) probes enhances the specificity of the assay by restricting mismatch hybridization and supporting accurate mutation detection9,10,11. Here, we describe our methods of sample processing, cfDNA extraction, preamplification of target alleles, dPCR and data analysis.

<table><tbody><tr><td>10 mM Tris-HCl, 0.1 mM EDTA DNA Suspension Buffer (pH 8.0)</td><td>Teknova</td><td>T0227</td><td></td></tr><tr><td>BD Vacutainer K2-EDTA 7.2 mg Blood Collection Tubes (10 mL)</td><td>Becton Dickinson Diagnostics</td><td>367525</td><td>For plasma collection</td></tr><tr><td>BD Vacutainer Plastic Serum tube with Red BD Hemogard Closure (10 mL)</td><td>Becton Dickinson Diagnostics</td><td>367895</td><td>For serum collection</td></tr><tr><td>Bleach</td><td>General Lab Supplier</td><td></td><td></td></tr><tr><td>Buffer ATL</td><td>Qiagen</td><td>19076</td><td></td></tr><tr><td>Cell-Free DNA Blood Collection Tubes</td><td>Streck</td><td>218961</td><td>cfDNA blood collection tubes (optional)</td></tr><tr><td>CentriVap Concentrator</td><td>Labconco</td><td>7810010</td><td></td></tr><tr><td>Forward Primer</td><td>Integrated DNA Technologies, Inc.</td><td></td><td>Custom design</td></tr><tr><td>MiniAmp Thermal Cycler</td><td>Thermofisher Scientific</td><td>A37834</td><td></td></tr><tr><td>Molecular Biology Grade Absolute Ethanol (200 proof)</td><td>General Lab Supplier</td><td></td><td></td></tr><tr><td>Mutant Probe</td><td>Integrated DNA Technologies, Inc.</td><td></td><td>Custom design</td></tr><tr><td>PAXgene Blood ccfDNA tubes</td><td>PreAnalytiX</td><td>768115</td><td>cfDNA blood collection tubes (optional)</td></tr><tr><td>PCR 8 well strip tube caps</td><td>VWR</td><td>10011-786</td><td></td></tr><tr><td>PCR 8 well strip tubes</td><td>Axygen</td><td>PCR-0208-C</td><td></td></tr><tr><td>Pipette (p20, p200, p1000)</td><td>General Lab Supplier</td><td></td><td></td></tr><tr><td>Pipette filter tips (p20, p200, p1000)</td><td>General Lab Supplier</td><td></td><td></td></tr><tr><td>Q5 Hot Start High-Fidelity 2x Master Mix</td><td>New England Biolabs Inc</td><td>M0494S</td><td></td></tr><tr><td>QIAamp Circulating Nucleic Acid HandBook&amp;nbsp;</td><td>Qiagen</td><td></td><td>cfDNA extraction kit handbook (2013 edition)</td></tr><tr><td>QIAamp Circulating Nucleic Acid Kit</td><td>Qiagen</td><td>55114</td><td>cfDNA extraction kit (Protocol Step 4)&amp;nbsp;</td></tr><tr><td>QIAamp MinElute Virus Spin Kit</td><td>Qiagen</td><td>57704</td><td>Optional kit for DNA extraction from small (&amp;lt; or =200 &amp;micro;L) sample volumes</td></tr><tr><td>Qiavac 24 Plus</td><td>Qiagen</td><td>19413</td><td>For use with QIAamp Circulating Nucleic Acids kit</td></tr><tr><td>Raindance Consumable Kit</td><td>Bio-Rad</td><td>20-04411</td><td>Contains droplet-generating instrument chips, quantification instrument chips, PCR strip caps including high-speed caps, and droplet stabilizing oil</td></tr><tr><td>Raindance Sense Instrument</td><td>Bio-Rad</td><td></td><td>Quantification instrument (used in Protocol Step 9)</td></tr><tr><td>Raindance Source Instrument</td><td>Bio-Rad</td><td></td><td>Droplet-generating instrument (used in Protocol Step 9)</td></tr><tr><td>RainDrop Analyst II Software</td><td>RainDance Technologies</td><td></td><td>Analyst software used for Data Analysis (Protocol Step 10)</td></tr><tr><td>Refrigerated Vapor Trap</td><td>Savant</td><td>RVT5105-115</td><td></td></tr><tr><td>Reverse Primer</td><td>Integrated DNA Technologies, Inc.</td><td></td><td>Custom design</td></tr><tr><td>Smartblock 2 mL</td><td>Eppendorf</td><td>05 412 506</td><td></td></tr><tr><td>TaqMan Genotyping Master Mix</td><td>Thermofisher Scientific</td><td>4371353</td><td></td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>14 285 562PM</td><td></td></tr><tr><td>WT Probe</td><td>Integrated DNA Technologies, Inc.</td><td></td><td>Custom design</td></tr></tbody></table>

detection and monitoring of tumor associated circulating dna in patient biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Figure 1&#160;shows representative results for successful detection of H3F3A p.K27M mutation in preamplified plasma (top left panel) and CSF (top right panel) cfDNA from two children with DMG. cfDNA samples were analyzed in replicate but only one representative graph is shown for each sample type. The dPCR plots show successful droplet generation, with 7-9 million droplets per PCR well (most of which are negative droplets that do not contain target DNA). A minimum of 7 million droplets per PCR well indicates a successful dropletization, while fewer than 7 million indicate failure of the assay, in which case the user should not proceed with data analysis. Figure 1 shows a clear separation between mutant and wildtype clusters along the x and y-axes, respectively. Robust wildtype clusters indicate that the cfDNA extraction was successful because template DNA is present. Mutant clusters show a MAF of 1.60% and 39.92% for the plasma and CSF samples, respectively, demonstrating positive detection of the mutation. For these patients, dPCR results are in accordance with tumor mutation status, as confirmed by genomic analysis of biopsy tumor tissue8. The negative control (bottom left panel) shows 0 mutant and 0 wildtype droplets, indicating that there was no contamination of the PCR reaction mixture. The positive control tumor tissue gDNA (bottom right panel) shows the mutation being detected at the expected allelic frequency for the particular tumor sample selected.&#160;
In Figure 2, representative results are shown for unsuccessful detection of the mutation of interest, H3F3A p.K27M, in plasma cfDNA from a child with DMG. Mutation status was confirmed by genomic analysis of tumor tissue8. Representative results from two replicate PCR wells are shown (top panels). The total number of droplets, including negative droplets, is between 7-9 million per PCR well, indicating successful dropletization. The wildtype clusters, plotted along the y-axis, show approximately 7-8,000 wildtype droplets per PCR well for the plasma cfDNA, indicating successful cfDNA extraction (as there is target wildtype DNA in the sample). However, 0 mutant droplets are detected in the plasma sample. The bottom left panel shows negative control (MB H2O) with no wildtype or mutant droplets; and the bottom right panel shows the positive control preamplified tumor tissue gDNA (0.025 ng) with positive mutation detection. As shown in this figure, the absence of mutation detection in plasma may not necessarily mean the patient is wildtype for the mutation of interest, as false negatives do occur8. In some cases, when the mutation is missed in the plasma collected at upfront biopsy, it is detected in the plasma obtained from the same patient at a later time point8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59721/59721fig1v2.jpg" /> 
Figure 1: Successful detection of H3F3A p.K27M mutation in preamplified plasma and CSF cfDNA from children with midline gliomas. <a href="https://www.jove.com/files/ftp_upload/59721/59721fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59721/59721fig2.jpg" /> 
Figure 2: False negative results obtained from analysis of a preamplified plasma cfDNA sample from a patient known to harbor the mutation of interest, H3F3A p.K27M, in the tumor. <a href="https://www.jove.com/files/ftp_upload/59721/59721fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids at JoVE.com

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

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Cancer Research

8.6K Views.  Children's National Health System. Dit protocol maakt de detectie van tumor geassocieerde mutaties en momenteel complimenten invasieve chirurgische ingrepen. Een andere kracht is de mogelijkheid om dit protocol te gebruiken om tumormutatie belasting overuren te controleren. De techniek maakt tumor genotyping zonder dat de werkelijke chirurgische weefsel biopsie die vooral nuttig is voor tumoren op uitdagende locaties, zoals de hersenstam met beperkte toegang tot weefsel.Bij afwezigheid van herhaalde biopsie zijn longitu...

Video: Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

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