Sensitive Quantification of Cell-Free Tumor DNA for Early Detection of Recurrence in Colorectal Cancer

The detection of plasma cell–free tumor DNA (ctDNA) is prognostic in colorectal cancer (CRC) and has potential for early prediction of disease recurrence. In clinical routine, ctDNA-based diagnostics are limited by the low concentration of ctDNA and error rates of standard next-generation sequencing (NGS) approaches. We evaluated the potential to increase the stability and yield of plasma cell–free DNA (cfDNA) for routine diagnostic purposes using different blood collection tubes and various manual or automated cfDNA extraction protocols. Sensitivity for low-level ctDNA was measured in KRAS-mutant cfDNA using an error-reduced NGS procedure. To test the applicability of rapid evaluation of ctDNA persistence in clinical routine, we prospectively analyzed postoperative samples of 67 CRC (stage II) patients. ctDNA detection was linear between 0.0045 and 45%, with high sensitivity (94%) and specificity (100%) for mutations at 0.1% VAF. The stability and yield of cfDNA were superior when using Streck BCT tubes and a protocol by Zymo Research. Sensitivity for ctDNA increased 1.5-fold by the integration of variant reads from triplicate PCRs and with PCR template concentration. In clinical samples, ctDNA persistence was found in ∼9% of samples, drawn 2 weeks after surgery. Moreover, in a retrospective analysis of 14 CRC patients with relapse during adjuvant therapy, we successfully detected ctDNA (median 0.38% VAF; range 0.18–5.04% VAF) in 92.85% of patients significantly prior (median 112 days) to imaging-based surveillance. Using optimized pre-analytical conditions, the detection of postoperative ctDNA is feasible with excellent sensitivity and allows the prediction of CRC recurrence in routine oncology testing.

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ThE uSE of AbErrAnT METhylATEd gEnES SEPT9 And VIM for clInIcAl dIAgnoSIS of colorEcTAl cAncEr

Clinical Practice ◽

10.17816/clinpract7415-20 ◽

2016 ◽

Vol 7 (4) ◽

pp. 15-20

Author(s):

O I Brovkina ◽

M G Gordiev ◽

D S Khodyrev ◽

A G Nikitin ◽

A V Averyanov

Keyword(s):

Colorectal Cancer ◽

High Sensitivity ◽

Test System ◽

Aberrant Methylation ◽

Adjacent Tissue ◽

Epigenetic Events ◽

Combined Use ◽

Ras Family ◽

Definition Of ◽

Tumor Dna

Definition of epigenetic disorders is important for early diagnosis of colorectal cancer. To obtain a model of diagnostic test system with high sensitivity and specificity, we determined the frequency of methylation in SEPT9 and VIM genes. Epigenetic events also were compared with mutations in the RAS family genes. It was confirmed the presence of aberrant methylation in SEPT9 and VIM genes in tumor cells. DNA of tumor samples was significantly more methylated than samples with DNA from adjacent tissue (P = 8,67E-19 for SEPT9 gene and P=8,68E-19 for VIM gene). In the group of patients carried mutations in KRAS or NRAS genes tumor DNA significantly more methylated in gene SEPT9 (P = 0.0018), in contrast to the tumor DNA from patients not carried mutations. We have demonstrated that the combined use of methylation markers can improve the sensitivity of the test systems used in the diagnostics of colon cancer.

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Mutation profiling of tumor DNA from plasma and tumor tissue of colorectal cancer patients with a novel, high-sensitivity multiplexed mutation detection platform

Oncotarget ◽

10.18632/oncotarget.3041 ◽

2014 ◽

Vol 6 (4) ◽

pp. 2549-2561 ◽

Cited By ~ 57

Author(s):

Evelyn Kidess ◽

Kyra Heirich ◽

Matthew Wiggin ◽

Valentina Vysotskaia ◽

Brendan C. Visser ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Patients ◽

Tumor Tissue ◽

Mutation Detection ◽

High Sensitivity ◽

Colorectal Cancer Patients ◽

Tumor Dna ◽

Mutation Profiling

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Plasma cell-free DNA and fraction of circulating KRAS mutations as prognostic in patients with metastatic colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.3_suppl.490 ◽

2014 ◽

Vol 32 (3_suppl) ◽

pp. 490-490 ◽

Cited By ~ 2

Author(s):

David Sefrioui ◽

Nasrin Vasseur ◽

Richard Sesboüé ◽

France Blanchard ◽

Alice Oden-Gangloff ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Plasma Cell ◽

Circulating Tumor Dna ◽

Dna Fragments ◽

Wild Type ◽

Kras Mutations ◽

Cell Free Dna ◽

Free Dna ◽

Tumor Dna

490 Background: It has been suggested that detection of circulating tumor DNA may be relevant in patients with metastatic colorectal cancer (mCRC). The main objective of the present study was to evaluate a method based on the TaqMan Mutation Detection Assay (TMDA) for the detection of circulating KRAS mutations in mCRC patients. Moreover, we also investigated the prognostic impact of the plasma cell-free DNA and the fraction of circulating KRAS mutations. Methods: The study was conducted from April to July 2013 and plasma samples were prospectively collected in a series of 35 mCRC patients treated with chemotherapy (CT). QIAamp Circulating Nucleic Acid kit was used for DNA extraction and Quant-iT High Sensitivity dsDNA Assay for cf-DNA quantification. Detection of circulating tumor DNA was based on the KRAS mutations detected in tumour and was performed in plasma by the castPCR Technology TMDA. Response to CT was assessed according to RECIST criteria. The results of plasma cf-DNA and level of mutant DNA fragments were correlated with response and 3-months survival. Results: We isolated and quantified plasma cf-DNA in all patients with a mean concentration of 106 ng/mL. Among them, 18 were wild-type and 17 mutated for KRAS in the tumour. Detection of circulating KRAS mutations was performed with TMDA in 23 patients (10 KRAS wild-type and 13 KRAS mutated). The sensitivity was 62% (8/13) and specificity 100% (0/10) with a level of circulating mutant DNA fragments ranging from 0 to 29%. Plasma cf-DNA and level of circulating mutant DNA were both significantly correlated with the 3-months survival (mean 36 versus 524 ng/mL, p=0.0015 and 2% versus 29%, p<0.0001). There was a non significant trend for response to CT (respectively p=0.14 and p=0.12). Conclusions: TMDA method is a simple, accurate and non-invasive tool for the detection of circulating tumor DNA. Our preliminary results also suggest that plasma cf-DNA and fraction of mutant DNA fragments could be prognostic markers in mCRC patients.

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Postoperative circulating tumor DNA as markers of recurrence risk in stages II to III colorectal cancer

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01089-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Gong Chen ◽

Junjie Peng ◽

Qian Xiao ◽

Hao-Xiang Wu ◽

Xiaojun Wu ◽

...

Keyword(s):

Colorectal Cancer ◽

Standard Of Care ◽

Recurrence Risk ◽

Disease Recurrence ◽

Circulating Tumor Dna ◽

Stage Ii ◽

Sampling Point ◽

Radiological Imaging ◽

Definitive Therapy ◽

Tumor Dna

Abstract Background Precise methods for postoperative risk stratification to guide the administration of adjuvant chemotherapy (ACT) in localized colorectal cancer (CRC) are still lacking. Here, we conducted a prospective, observational, and multicenter study to investigate the utility of circulating tumor DNA (ctDNA) in predicting the recurrence risk. Methods From September 2017 to March 2020, 276 patients with stage II/III CRC were prospectively recruited in this study and 240 evaluable patients were retained for analysis, of which 1290 serial plasma samples were collected. Somatic variants in both the primary tumor and plasma were detected via a targeted sequencing panel of 425 cancer-related genes. Patients were treated and followed up per standard of care. Results Preoperatively, ctDNA was detectable in 154 of 240 patients (64.2%). At day 3–7 postoperation, ctDNA positivity was associated with remarkably high recurrence risk (hazard ratio [HR], 10.98; 95%CI, 5.31–22.72; P < 0.001). ctDNA clearance and recurrence-free status was achieved in 5 out of 17 ctDNA-positive patients who were subjected to ACT. Likewise, at the first sampling point after ACT, ctDNA-positive patients were 12 times more likely to experience recurrence (HR, 12.76; 95%CI, 5.39–30.19; P < 0.001). During surveillance after definitive therapy, ctDNA positivity was also associated with extremely high recurrence risk (HR, 32.02; 95%CI, 10.79–95.08; P < 0.001). In all multivariate analyses, ctDNA positivity remained the most significant and independent predictor of recurrence-free survival after adjusting for known clinicopathological risk factors. Serial ctDNA analyses identified recurrence with an overall accuracy of 92.0% and could detect disease recurrence ahead of radiological imaging with a mean lead time of 5.01 months. Conclusions Postoperative serial ctDNA detection predicted high relapse risk and identified disease recurrence ahead of radiological imaging in patients with stage II/III CRC. ctDNA may be used to guide the decision-making in postsurgical management.

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5 Multiple myeloma flow cytometry panel validated for clinical monitoring of patients

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0005 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A5-A5

Author(s):

Bevan Gang ◽

Vicky Sgouroudis ◽

Virginia Litwin ◽

Anita Boyapati

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Plasma Cell ◽

Plasma Cells ◽

Temperature Stability ◽

Clinical Samples ◽

Blood Collection ◽

Significant Heterogeneity ◽

Flow Cytometric ◽

Limit Of Quantitation

BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy with significant heterogeneity in clinical presentation. Plasma cells are antibody-producing cells of lymphoid origin that are resident in secondary lymphoid organs and in the bone marrow (BM). The detection of circulating malignant plasma cells using flow cytometry has also been described in patients with MM. Enumerating and phenotyping malignant plasma cells in the BM and peripheral blood (PB) may be of value when evaluating the presence of MM antigens targeted by therapies before and during treatment and at relapse. To this end, a flow cytometric panel was developed to enumerate and characterize malignant plasma cells and additional immune subsets.MethodsPB and BM aspirates (BMA) were obtained from healthy donors and MM donors who consented to research testing. MM cell lines were also used to spike into donor samples to detect specific antigens (collected in Cyto-Chex® blood collection tubes). Samples were then transferred to TruCount tubes to enumerate immune populations. Fluorescently labeled antibodies directed against CD38, CD138, CD56, CD45, BCMA were evaluated to assess parameters such as time and temperature stability of the reportable immune populations by monitoring the frequencies of the populations. In addition, the limit of quantitation, intra- and inter-assay precision were determined.ResultsThe MM Counting Panel was optimized to leverage antigen expression and fluorophore combinations. A gating strategy enabled enumeration of MM cells based on antigens that can be further subdivided based on BCMA expression. Further testing showed that the precision in frequencies and absolute counts of key reportable populations was deemed acceptable (%CV of <30%). The precision was within the acceptance criteria of%CV <30% for populations with =100 cells. Stability testing revealed that samples were more stable at ambient temperature relative to 4oC, with stability being maintained for 48 h post-collection, where at least 85% of reportable immune readouts were stable (%change <30% relative to baseline), for BMA and PB from various donors (healthy and MM).The panel was ultimately deployed for use with clinical samples from MM clinical trials. Clinical data generated from the MM Counting Panel allowed the identification of malignant plasma cell populations in BMA of patients from trial assessing a BCMAxCD3 bi-specific antibody (NCT03761108).ConclusionsA flow cytometric assay to enumerate and identify normal and malignant plasma cells in MM patients was successfully developed. The approach used can be applied to develop assays for other indications in which patients are treated with therapies.

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The Latin Square Task as a Measure of Relational Reasoning

European Journal of Psychological Assessment ◽

10.1027/1015-5759/a000520 ◽

2020 ◽

Vol 36 (2) ◽

pp. 296-302 ◽

Cited By ~ 1

Author(s):

Luke J. Hearne ◽

Damian P. Birney ◽

Luca Cocchi ◽

Jason B. Mattingley

Keyword(s):

Brain Imaging ◽

Response Times ◽

Latin Square ◽

Error Rates ◽

Functional Brain Imaging ◽

Inspection Time ◽

Relational Reasoning ◽

Validity And Reliability ◽

The Stability ◽

Test Retest Reliability

Abstract. The Latin Square Task (LST) is a relational reasoning paradigm developed by Birney, Halford, and Andrews (2006) . Previous work has shown that the LST elicits typical reasoning complexity effects, such that increases in complexity are associated with decrements in task accuracy and increases in response times. Here we modified the LST for use in functional brain imaging experiments, in which presentation durations must be strictly controlled, and assessed its validity and reliability. Modifications included presenting the components within each trial serially, such that the reasoning and response periods were separated. In addition, the inspection time for each LST problem was constrained to five seconds. We replicated previous findings of higher error rates and slower response times with increasing relational complexity and observed relatively large effect sizes (η2p > 0.70, r > .50). Moreover, measures of internal consistency and test-retest reliability confirmed the stability of the LST within and across separate testing sessions. Interestingly, we found that limiting the inspection time for individual problems in the LST had little effect on accuracy relative to the unconstrained times used in previous work, a finding that is important for future brain imaging experiments aimed at investigating the neural correlates of relational reasoning.

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