Automated DNA extraction using cellulose magnetic beads can improve <i>EGFR</i> point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

Nakashima, Chiho; Sato, Akemi; Abe, Tomonori; Kato, Junichi; Hirai, Mitsuharu; Nakamura, Tomomi; Komiya, Kazutoshi; Sueoka, Eisaburo; Kimura, Shinya; Sueoka‐Aragane, Naoko

doi:10.18632/oncotarget.25388

Cited by 28 publications

(28 citation statements)

References 31 publications

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“…Chan, K. C. et al reported that repeated freeze-thaw cycles lead to fragmentation of cfDNA even if the DNA concentration does not change [ 34 ]. We recently demonstrated that cfDNA integrity is maintained after isolation using a silica membrane spin column [ 22 ]. This suggests that fragmentation of DNA occurs during long-term storage, leading to difficulty conducting PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, pre-analytical procedures, including type of blood collection tube, choice of anticoagulant, centrifugation protocol, storage conditions, and DNA extraction method, should be reviewed. We recently reported that DNA extraction with cellulose magnetic beads produced higher recovery of cfDNA and better quality than with a silica membrane spin column system, leading to an improved efficiency of EGF R mutation detection [ 22 ]. However, assessment of other steps of pre-analytical procedures has not been made.…”

Section: Introductionmentioning

confidence: 99%

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Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

et al. 2018

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Liquid biopsy with circulating free DNA (cfDNA) is a recommended alternative method of re-biopsy. Quality control with cfDNA is indispensable for precise examinations, and it is desirable to achieve high-quality cfDNA separation. We investigated two issues: the influence of pre-analytical procedures on cfDNA analysis performed as a routine procedure in a standard clinical laboratory, and the extent of deterioration of cfDNA quality due to long-term storage. Comparisons among blood collection tube types, storage temperatures, and periods of blood separation were performed in terms of cfDNA quantification, cfDNA size distribution, and detection of EGFR mutations. Quality of cfDNA was better with collection tubes containing 3.2% sodium citrate than with those containing EDTA 2K, and was maintained with storage at 4° C for up to 72 h after blood collection, equivalent to results with cell-stabilizing blood collection tubes. Analysis of cfDNA stored for 7 years showed that samples with low allele frequency (AF) deteriorated more readily than samples with high AF. Despite the same storage period and extraction method, AF of plasma stored for 7 years was remarkably lower than that of cfDNA. However, deterioration due to long-term plasma storage was overcome by changing the DNA extraction method from a silica membrane spin column to a cellulose magnetic beads system. These results can guide the establishment of standardized pre-analytical procedures for liquid biopsy with cfDNA.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

et al. 2018

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“…Circulating free DNA (cfDNA) exists in peripheral blood and is derived from tumor cells and from normal cells (mainly lymphocytes). Recently, we reported that cfDNA was better isolated with cellulose magnetic beads for DNA capturing, and the amount of ctDNA was greater than that obtained with silica membrane spin columns [7]. Using the capillary electrophoresis system, we also demonstrated the existence of bimodal peaks, comprising long fragment cfDNA of 5Kb and short fragment cfDNA of 170bp.…”

Section: Introductionmentioning

confidence: 79%

Origin of circulating free DNA in patients with lung cancer

et al. 2020

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Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.

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“…Age prevalence of the tumor is among second and third decades [ 8 ]. Symptoms of the patients with JGCT are headaches, retinopathy, double vision, dizziness, nausea, vomiting, polyuria, and proteinuria [ 7 ]. Macroscopically, the tumor is yellow to tan and has fibrous capsule.…”

Section: Discussionmentioning

confidence: 99%

“…Gene mutations were detected by mutation-biased PCR and quenching probe (MBP-QP) system using i-densy (IS-5320, ARKRAY Inc., Kyoto, Japan) [ 7 ]. DNA was extracted from 3 pieces of 5 μ m paraffin embedded tissue sample by using Maxwell 16® system (Promega Corporation, Tokyo, Japan) after proteinase K treatment (70°C, overnight).…”

Section: Methodsmentioning

confidence: 99%

Case Report of Atypical Juxtaglomerular Cell Tumor

Munakata

Tomiyama

Takayama

2018

Case Reports in Pathology

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Juxtaglomerular cell tumor (JGCT) is a rare renal tumor, producing renin and behaving almost in a benign fashion. So far, only three cases have been reported as malignant. We report a rare case with atypical JGCT. A 74-year-old male was referred to our hospital due to hypertension, proteinuria, and hematuria. Abdominal CT revealed a mass measured in 9.7×7.0 cm in the lower portion of the right kidney. Right kidney was removed laparoscopically. Grossly, white to tan tumor with massive hemorrhage and necrosis occupied the lower portion of the right kidney. Microscopically, tumor grew in a solid fashion. Tumor cells were polygonal to ovoid cells with round nuclei and clear to eosinophilic cytoplasm. Mitosis was found in 5 per 10 HPF. Immunohistochemically, tumor cells were stained by vimentin and CD34. Some tumor cells were also positive for renin. Electron micrograph showed near rhomboid crystalline structure in the tumor cells. Because of massive necrosis and mitotic figures, diagnosis of atypical (potentially malignant) JGCT was rendered. Gene mutations for IDH1, PIK3CA, K-ras, N-ras, Braf, and EGFR were not found by MBP-QP system.

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Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

Cited by 28 publications

References 31 publications

Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Origin of circulating free DNA in patients with lung cancer

Case Report of Atypical Juxtaglomerular Cell Tumor

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