Evaluation of a Liquid Biopsy Protocol using Ultra-Deep Massive Parallel Sequencing for Detecting and Quantifying Circulation Tumor DNA in Colorectal Cancer Patients

Nguyen, Hung; Tran, Duc Huy; Ngo, Quoc Dat; Pham, Hong-Anh Thi; Tran, Thuy Thi Thu; Tran, Van Thi Thanh; Pham, Truong-Vinh Ngoc; Le, Trung Kien; Le, Ngoc-An Trinh; Nguyễn, Như Gia; Vo, Binh Thanh; Nguyen, Luan Thanh; Nguyen, Thien-Chi Van; Bui, Quynh Tram Nguyen; Nguyen, Huu-Nguyen; Luong, Bac An; Le, Linh-Thy; Duc, Minh; Do, Thanh-Thuy Thi; Hoang, Anh Vu; Dinh, Kiet Truong; Phan, Minh-Duy; Tran, Le Son; Giang, Hoa; Nguyen, Hoai-Nghia

doi:10.1080/07357907.2020.1713350

Cited by 21 publications

(16 citation statements)

References 44 publications

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“…Another 6 studies also used commercial NGS panel for BRAF mutation testing in plasma sample. Nguyen et al [ 26 ] used commercial xGen predesigned gene capture pools (Integrated DNA Technologies) and obtained complete agreement of BRAF mutation results between plasma and tumor tissue sample from 50 CRC patients. Diefenbach et al [ 27 ] used whole exome sequencing panel (SureSelect, Agilent) in 10 melanoma patients and the calculated sensitivity and specificity were 66.7% and 100%, respectively, with concordance rate at 80%.…”

Section: Resultsmentioning

confidence: 99%

Reliability of BRAF mutation detection using plasma sample

Cai

Xie

et al. 2021

Medicine

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Background: Testing of B-Raf proto-oncogene ( BRAF ) mutation in tumor is necessary before targeted therapies are given. When tumor samples are not available, plasma samples are commonly used for the testing of BRAF mutation. The aim of this study was to investigate the diagnostic accuracy of BRAF mutation testing using plasma sample of cancer patients. Methods: Databases of Pubmed, Embase, and Cochrane Library were searched for eligible studies investigating BRAF mutation in paired tissue and plasma samples of cancer patients. A total of 798 publications were identified after database searching. After removing 229 duplicated publications, 569 studies were screened using the following exclusion criteria: (1) BRAF mutation not measured in plasma or in tumor sample; (2) lacking BRAF -wildtype or BRAF -mutated samples; (3) tissue and plasma samples not paired; (4) lacking tumor or plasma samples; (5) not plasma sample; (6) not cancer; (7) un-interpretable data. Accuracy data and relevant information were extracted from each eligible study by 2 independent researchers and analyzed using statistical software. Results: After pooling the accuracy data from 3943 patients of the 53 eligible studies, the pooled sensitivity, specificity, and diagnostic odds ratio of BRAF mutation testing using plasma sample were 69%, 98%, and 55.78, respectively. Area under curve of summary receiver operating characteristic curve was 0.9435. Subgroup analysis indicated that BRAF mutation testing using plasma had overall higher accuracy (diagnostic odds ratio of 89.17) in colorectal cancer, compared to melanoma and thyroid carcinoma. In addition, next-generation sequencing had an overall higher accuracy in detecting BRAF mutation using plasma sample (diagnostic odds ratio of 63.90), compared to digital polymerase chain reaction (PCR) and conventional PCR, while digital PCR showed the highest sensitivity (74%) among the 3 techniques. Conclusion: BRAF testing using plasma sample showed an overall high accuracy compared to paired tumor tissue sample, which could be used for cancer genotyping when tissue sample is not available. Large prospective studies are needed to further investigate the accuracy of BRAF mutation testing in plasma sample.

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Section: Resultsmentioning

confidence: 99%

Reliability of BRAF mutation detection using plasma sample

Cai

Xie

et al. 2021

Medicine

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“…Following sequencing, an average of 38 million reads were obtained from the three patient libraries, with 35-42% of reads mapping to target regions (Supplementary Table 2). This low on-target rate was not unexpected and has been observed with small hybrid capture sequencing panels with low input DNA, impacting both assay cost-efficacy and sensitivity [17,18]. We hypothesized that the on-target rate could be improved by two further modifications to the protocol: first, increasing the post-capture washes (four washes with 400 μl WB2 for 20 min each) to remove mismatched and thus weakly binding library fragments; and second, performing dCAP-Seq (a second capture for 5 h followed by a further five cycles of amplification) to further enrich for targeted libraries [9].…”

Section: Assay Optimizationmentioning

confidence: 63%

Analytical Validation of an Error-Corrected Ultra-Sensitive Ctdna Next-Generation Sequencing Assay

et al. 2020

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Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (<1%), with high concordance observed between the in-house and commercial assays. The optimized protocol dramatically improved the efficiency of the assay and enabled the detection of low-frequency somatic variants from plasma cell-free DNA (cfDNA).

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“…Moreover, the test involves many washing and incubation steps with a total analysis time of about 2 h [ 48 ]. Compared to the previously reported biosensors for ctDNA detection in real samples ( Table S3 ) [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ], the advantages of the proposed multi-plex test are related mostly to the cost, run time, and simplicity, as well as the universality and advanced multiplexing potential.…”

Section: Discussionmentioning

confidence: 93%

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

et al. 2022

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In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

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Evaluation of a Liquid Biopsy Protocol using Ultra-Deep Massive Parallel Sequencing for Detecting and Quantifying Circulation Tumor DNA in Colorectal Cancer Patients

Cited by 21 publications

References 44 publications

Reliability of BRAF mutation detection using plasma sample

Reliability of BRAF mutation detection using plasma sample

Analytical Validation of an Error-Corrected Ultra-Sensitive Ctdna Next-Generation Sequencing Assay

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

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