Impact of DNA integrity on the success rate of tissue‐based next‐generation sequencing: Lessons from nationwide cancer genome screening project SCRUM‐Japan GI‐SCREEN

A sufficiently large tissue sample is required to perform next-generation sequencing (NGS) with a high success rate, but the majority of patients with advanced non-small-cell lung cancer (NSCLC) are diagnosed with small biopsy specimens. Biopsy samples were collected from 184 patients with bronchoscopically diagnosed NSCLC. The tissue surface area, tumor cell count, and tumor content rate of each biopsy sample were evaluated. The impact of the cut-off criteria for the tissue surface area (≥1 mm2) and tumor content rate (≥30%) on the success rate of the Oncomine Dx Target Test (ODxTT) was evaluated. The mean tissue surface area of the transbronchial biopsies was 1.23 ± 0.85 mm2 when small endobronchial ultrasonography with a guide sheath (EBUS-GS) was used, 2.16 ± 1.49 mm2 with large EBUS-GS, and 1.81 ± 0.75 mm2 with endobronchial biopsy (EBB). The proportion of samples with a tissue surface area of ≥1 mm2 was 48.8% for small EBUS-GS, 79.2% for large EBUS-GS, and 78.6% for EBB. Sixty-nine patients underwent ODxTT. The success rate of DNA sequencing was 84.1% and that of RNA sequencing was 92.7% over all patients. The success rate of DNA (RNA) sequencing was 57.1% (71.4%) for small EBUS-GS (n = 14), 93.4% (96.9%) for large EBUS-GS (n = 32), 62.5% (100%) for EBB (n = 8), and 100% (100%) for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n = 15). Regardless of the device used, a tissue surface area of ≥ 1 mm2 is adequate for samples to be tested with NGS.

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Characterising Somatic Mutations in Cancer Genome by Means of Next-generation Sequencing

Encyclopedia of Life Sciences ◽

10.1002/9780470015902.a0023379 ◽

2012 ◽

Author(s):

Mei Ling Chong ◽

Chee Seng Ku ◽

Mengchu Wu ◽

Richie Soong

Keyword(s):

Next Generation Sequencing ◽

Somatic Mutations ◽

Cancer Genome ◽

Next Generation ◽

Generation Sequencing

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Tumor BRCA Test for Patients with Epithelial Ovarian Cancer: The Role of Molecular Pathology in the Era of PARP Inhibitor Therapy

Cancers ◽

10.3390/cancers11111641 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1641 ◽

Cited By ~ 5

Author(s):

Caterina Fumagalli ◽

Federica Tomao ◽

Ilaria Betella ◽

Alessandra Rappa ◽

Mariarosaria Calvello ◽

...

Keyword(s):

Ovarian Cancer ◽

Next Generation Sequencing ◽

Epithelial Ovarian Cancer ◽

Success Rate ◽

Parp Inhibitor ◽

Turnaround Time ◽

Molecular Diagnostic ◽

Next Generation ◽

Clinical Implementation ◽

Generation Sequencing

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

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Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples

Clinical Chemistry ◽

10.1373/clinchem.2012.193409 ◽

2013 ◽

Vol 59 (5) ◽

pp. 815-823 ◽

Cited By ~ 71

Author(s):

Audrey Didelot ◽

Steve K Kotsopoulos ◽

Audrey Lupo ◽

Deniz Pekin ◽

Xinyu Li ◽

...

Keyword(s):

Next Generation Sequencing ◽

Simultaneous Detection ◽

Digital Pcr ◽

Clinical Samples ◽

Dna Integrity ◽

Next Generation ◽

Sequencing Data ◽

Integrity Test ◽

Picoliter Droplet ◽

Generation Sequencing

BACKGROUND Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet–based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.

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