Statistical analysis of mutant allele frequency level of circulating cell-free DNA and blood cells in healthy individuals

Xia, Ligang; Li, Zhoufang; Zhou, Bo; Tian, Geng; Zeng, Lidong; Dai, Hongyu; Li, Xiaohua; Liu, Chao-Yu; Lu, Songhe; Xu, Feiyue; Tu, Xiaonian; Deng, Fang; Xie, Yu; Huang, Weiren; He, Jiankui

doi:10.1038/s41598-017-06106-1

Cited by 43 publications

(36 citation statements)

References 27 publications

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“…29 The mutant allele frequencies in cfDNA from apoptotic cells were almost identical with those in the blood cells. 29,30 Consistent with this, we observed that mutant allele frequencies in cfDNA were distinct from gDNA, and that had a significant correlation with gDNA in coexisting mutations. Therefore, a significant fraction of alterations identified in cfDNA should be considered as the background mutations from apoptotic DNA fragments.…”

Section: Discussionsupporting

confidence: 83%

“…Fragments of circulating cfDNA were shown to be derived from apoptosis, necrosis or active secretion, which can be detected in the cell‐free components of plasma and serum . The mutant allele frequencies in cfDNA from apoptotic cells were almost identical with those in the blood cells . Consistent with this, we observed that mutant allele frequencies in cfDNA were distinct from gDNA, and that had a significant correlation with gDNA in coexisting mutations.…”

Section: Discussionsupporting

confidence: 80%

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Identification of driver genes and somatic mutations in cell‐free DNA of patients with pulmonary lymphangioleiomyomatosis

Zhang

Wang

et al. 2019

Intl Journal of Cancer

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Next‐generation sequencing of cell‐free circulating DNA (cfDNA) has emerged as promising technique for identifying minimally invasive genomic profiling of tumor cells recently. However, it remains relatively unknown in LAM disease. In our study, paired cfDNA and genomic DNA (gDNA) in blood samples were obtained from 23 LAM patients and seven healthy controls to explore mutations profiles of targeted 70 cancer‐related genes. As results, log2‐based allele frequencies of mutations in cfDNA were significantly different from those of gDNA. By comparing the mutual mutations identified both in cfDNA and gDNA, a significant correlation was also observed. After removing mutations in gDNA, distinct somatic mutation profiles of cfDNA were observed in LAM patients. Forty of 70 targeted genes had recurrent mutations, of which ATM, BRCA2 and APC showed the highest frequency. Based on the mutation, correlation network constructed of 40 mutated genes, 11 hub genes bearing intensive interactions were highlighted, including BRCA1, BRCA2, RAD50, RB1, NF1, APC, MLH3, ATM, PDGFRA, PALB2 and BLM. Expression of the hub genes showed significant clusters between LAM patients and controls and that RAD50 and BRCA2 had the strongest associations with subject phenotypes. Myogenesis and estrogen response were confirmed to be positively regulated in LAM patients. Collectively, our study provided a landscape of genomic alterations in LAM and discovered several potential driver genes, that is, BRCA2 and RAD50, which shed a substantial light on the clinical application of key molecular markers and potential therapy targets for precision diagnosis and treatment in the future.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 80%

Identification of driver genes and somatic mutations in cell‐free DNA of patients with pulmonary lymphangioleiomyomatosis

Zhang

Wang

et al. 2019

Intl Journal of Cancer

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“…The above collected DNA samples were analyzed by our proprietary Sec-Seq technique as described previously (17). Briefly, blood sample were processed to separate the plasma from blood cells by centrifugation.…”

Section: Sequencing Analysismentioning

confidence: 99%

Multi-Center Study of Resectable Lung Lesions by Ultra-Deep Sequencing of Targeted Genes in Plasma Cell-Free DNA to Assess Nodule Malignancy and Detect Lung Cancers

Xie

X²,

et al. 2018

Preprint

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BACKGROUND Early detection of lung cancer to allow curative treatment remains challenging.Cell-free circulating tumor DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery. METHODS The multi-center clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white blood cell genomic DNA (gDNA) and tumor tissue gDNA of each patient were analyzed by ultra-deep sequencing to an average of 35,000X of the coding regions of 65 lung cancer-related genes.RESULTS The cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients is at Stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%.When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for Stage I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harbored mutations in the lung cancer patients with the largest number found in TP53 (30% plasma and 62% tumor tissue samples) and EGFR (20% and 40%, respectively).CONCLUSION Plasma ctDNA was analyzed in lung nodule assessment and early cancer detection while an algorithm combining clinical information enhanced the test performance.

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“…In order to focus on the above questions, our previous studies explored new resin to increase the recovery rate in ctDNA extraction and examined the background somatic mutations originated from blood cells and cfDNA in 821 non-cancer individuals based on ultra-deep sequencing and we also described the baseline reference 18 . Now we are focusing on the understanding of ctDNA profiles in cancer patients after we developed an updated method called "Sec-Seq" with exogenous molecular barcoding and custom-probe capture to suppress the systematic errors and detect the genetic alterations including mutations, insertion and deletion in early stages of lung cancer.…”

Section: Introductionmentioning

confidence: 99%

The Early Diagnosis in Lung Cancer by the Detection of Circulating Tumor DNA

Tian

Xie

et al. 2017

Preprint

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Background Remarkable advances for clinical diagnosis and treatment in cancers including lung cancer involve cell-free circulating tumor DNA (ctDNA) detection through next generation sequencing. However, before the sensitivity and specificity of ctDNA detection can be widely recognized, the consistency of mutations in tumor tissue and ctDNA should be evaluated. The urgency of this consistency is extremely obvious in lung cancer to which great attention has been paid to in liquid biopsy field. Methods We have developed an approach named systematic error correction sequencing (Sec-Seq) to improve the evaluation of sequence alterations in circulating cell-free DNA. Averagely 10 ml preoperative blood samples were collected from 30 patients containing pulmonary space occupying pathological changes by traditional clinic diagnosis. cfDNA from plasma, genomic DNA from white blood cells, and genomic DNA from solid tumor of above patients were extracted and constructed as libraries for each sample before subjected to sequencing by a panel contains 50 cancer-associated genes encompassing 29 kb by custom probe hybridization capture with average depth >40000, 7000, or 6300 folds respectively. Results Detection limit for mutant allele frequency in our study was 0.1%. The sequencing results were analyzed by bioinformatic expertise based on our previous studies on the baseline mutation profiling of circulating cell-free DNA and the clinicopathological data of these patients. Among all the lung cancer patients, 78% patients were predicted as positive by ctDNA sequencing when the shreshold was defined as at least one of the hotspot mutations detected in the blood (ctDNA) was also detected in tumor tissue. Pneumonia and pulmonary tuberculosis were detected as negative according to the above standard. When evaluating all hotspots in driver genes in the panel, 24% mutations detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). When evaluating all genetic variations in the panel, including all the driver genes and passenger genes, 28% detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). Positive detection rates of plasma ctDNA in stage I lung cancer patients is 85%, compared with 17% of tumor biomarkers. Conclusion We demonstrated the importance of sequencing both circulating cell-free DNA and genomic DNA in tumor tissue for ctDNA detection in lung cancer currently. We also determined and confirmed the consistency of ctDNA and tumor tissue through NGS according to the criteria explored in our studies. Our strategy can initially distinguish the lung cancer from benign lesions of lung. Our work shows that the consistency will be benefited from the optimization in sensitivity and specificity in ctDNA detection.reuse allowed without permission.

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Statistical analysis of mutant allele frequency level of circulating cell-free DNA and blood cells in healthy individuals

Cited by 43 publications

References 27 publications

Identification of driver genes and somatic mutations in cell‐free DNA of patients with pulmonary lymphangioleiomyomatosis

Identification of driver genes and somatic mutations in cell‐free DNA of patients with pulmonary lymphangioleiomyomatosis

Multi-Center Study of Resectable Lung Lesions by Ultra-Deep Sequencing of Targeted Genes in Plasma Cell-Free DNA to Assess Nodule Malignancy and Detect Lung Cancers

The Early Diagnosis in Lung Cancer by the Detection of Circulating Tumor DNA

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