Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model

Maggi, Elaine C.; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K.; Vijg, Jan; Montagna, Cristina

doi:10.3389/fgene.2018.00006

Cited by 23 publications

(12 citation statements)

References 56 publications

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“…S1) 43 . The HMW DNA could be removed using purification beads 43 . Breast cancer 1 ccfDNA contains HMW DNA and it seems to be similar to healthy ccfDNA samples for the detection of CNVs and both CNV and SNV signatures for female healthy individual 1 (Figs.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Mauger

Horgues

Pierre-Jean

et al. 2020

Sci Rep

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circulating cell-free DnA (ccfDnA) has great potential for non-invasive diagnosis, prognosis and monitoring treatment of disease. However, a sensitive and specific whole-genome sequencing (WGS) method is required to identify novel genetic variations (i.e., SnVs, cnVs and inDeLS) on ccfDnA that can be used as clinical biomarkers. In this article, five WGS methods were compared: ThruPLEX Plasma-seq, QIAseq cfDNA All-in-One, NEXTFLEX Cell Free DNA-seq, Accel-NGS 2 S PCR FREE DNA and Accel-NGS 2 S PLUS DNA. The Accel PCR-free kit did not produce enough material for sequencing. The other kits had significant common number of SNVs, INDELs and CNVs and showed similar results for SnVs and cnVs. the detection of variants and genomic signatures depends more upon the type of plasma sample rather than the WGS method used. Accel detected several variants not observed by the other kits. ThruPLEX seemed to identify more low-abundant SNVs and SNV signatures were similar to signatures observed with the QIAseq kit. Accel and NEXTFLEX had similar CNV and SNV signatures. These results demonstrate the importance of establishing a standardized workflow for identifying noninvasive candidate biomarkers. Moreover, the combination of variants discovered in ccfDNA using WGS has the potential to identify enrichment pathways, while the analysis of signatures could identify new subgroups of patients. The analysis of circulating cell-free DNA (ccfDNA) from plasma bears great promise for diagnosis, prognosis and monitoring the treatment of cancer 1. In the context of precision medicine, the identification of novel non-invasive biomarkers is crucial but the analysis of ccfDNA is still a challenge. Indeed, ccfDNA is low concentrated, highly fragmented and the abundance depends on the type and the stage of cancer and the pre-analytical steps 2,3-5. Due to its properties, a complete workflow for sample preparation, library preparation, sequencing and data analysis should be performed to ensure standardization of sample analysis especially in the case of clinical cohorts 4,6,7. Pre-analytical steps including sample collection, storage, processing and extraction were compared to maximize the yield and size of ccfDNA 3,5,8-12. Furthermore, size analysis and quantification methods were used to evaluate the extracted ccfDNA. Sensitive approaches such as quantitative PCR, digital PCR, mass spectrometry and next generation sequencing (NGS) are commonly applied to analyze extracted ccfDNA 2. With the improvement of NGS analysis, whole-genome sequencing (WGS) is a great approach to identify all types of genomic alteration including single nucleotide variant (SNV), insertion and deletion (INDEL), copy number variation (CNV) and structural variant (SV) for the identification of candidate biomarkers in cancer 13. In particular, several specific and sensitive low-coverage sequencing approaches have been applied for the analysis of CNVs from cancer plasma samples 14-20. In addition, recent WGS studies allowed the analysis of nucleosome

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Section: Discussionmentioning

confidence: 99%

“…Furthermore, fragment size analysis is performed before sequencing to verify the size of ccfDNA and to ensure it does not contain HMW DNA which is about 10,000 bp ( Supplementary Fig. S1) 43 . The HMW DNA could be removed using purification beads 43 .…”

Section: Discussionmentioning

confidence: 99%

Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Mauger

Horgues

Pierre-Jean

et al. 2020

Sci Rep

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“…After eluting the samples in the provided elution buffer, the concentration of total cfDNA per mL of serum was calculated using uometric quanti cation (Qubit). Low-molecular weight tumor-related DNA (LMW cfDNA) was puri ed from contaminating high molecular weight genomic DNA using Solid Phase Reversible Immobilisation beads (SPRI) from Beckman Coulter (Agencourt AMPure cat#A63880) using methods previously described by our laboratory (8). This DNA puri cation step has been demonstrated to remove contaminating traces of high-molecular weight genomic DNA (~ 10,380 bp) from tumor-derived DNA (1 50-200 bp).…”

Section: Methodsmentioning

confidence: 99%

Low molecular weight serum cell-free DNA concentration is associated with clinicopathologic indices of poor prognosis in women with uterine cancer

Gressel

Maggi

Harmon

et al. 2020

Preprint

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Background Serum cell-free DNA (cfDNA) holds promise as a non-invasive cancer biomarker. The objective of this study was to evaluate the association of cfDNA concentration with clinicopathologic variables of poor prognosis and overall survival among women with uterine cancer compared to benign cancer-free controls. Methods cfDNA was extracted from the serum of 91 women with multiple uterine cancer histologies and 22 post-menopausal controls without cancer. Low molecular weight (LMW) cfDNA was separated from contaminating genomic high molecular weight cfDNA using paramagnetic bead purification and its concentration was measured using fluorometric quantification. Clinicopathologic data was abstracted from the electronic medical record. The association between serum cfDNA concentration, clinicopathologic variables, and overall survival was assessed using linear regression modelling, Cox proportional hazards modelling, and the Kaplan-Meier method. Results Median total serum cfDNA concentration for the cohort was 69.2 ng/mL (IQR 37.4, 132.3) and median LMW cfDNA concentration was 23.8 ng/mL (IQR 14.9, 44.4). There were no significant differences in total serum cfDNA concentration with any clinicopathologic variables. However, LMW cfDNA concentration was significantly higher in serum of women with cancer (25.8 ng/mL IQR 16.0, 49.6) compared to benign controls (15.5 ng/mL IQR 9.3, 25.8 ng/mL) (p <0.01). It is also significantly higher among women with early stage cancer than benign controls (p < 0.01). There were also significant associations between LMW cfDNA concentration and stage of cancer (p = 0.01) and histology (p = 0.02). Patients with leiomyosarcoma and carcinosarcoma had higher cfDNA concentrations than those with endometrioid cancer. Over a median follow-up of 51.9 months, 75th percentile for overall survival for women with cancer was 24.0 months. Higher LMW cfDNA concentrations is associated with lower survival among women with cancer (p < 0.01). Conclusions Serum LMW cfDNA concentration is associated with overall survival in women with uterine cancer, and it is higher among women with uterine cancer compared to those of controls.

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“…After eluting the samples in the provided elution buffer, the concentration of total cfDNA per mL of serum was calculated using fluometric quantification (Qubit). Low-molecular weight tumor-related DNA (LMW cfDNA) was purified from contaminating high molecular weight genomic DNA using Solid Phase Reversible Immobilisation beads (SPRI) from Beckman Coulter (Agencourt AMPure cat#A63880) using methods previously described by our laboratory [8]. This DNA purification step has been demonstrated to remove contaminating traces of high-molecular weight genomic DNA (~ 10,380 bp) from tumor-derived DNA (~ 150-200 bp).…”

Section: Cell-free Dna Isolation Purification and Quantificationmentioning

confidence: 99%

Low molecular weight serum cell-free DNA concentration is associated with clinicopathologic indices of poor prognosis in women with uterine cancer

et al. 2020

Self Cite

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Background: Serum cell-free DNA (cfDNA) holds promise as a non-invasive cancer biomarker. The objective of this study was to evaluate the association of cfDNA concentration with clinicopathologic variables of poor prognosis and overall survival among women with uterine cancer compared to benign cancer-free controls. Methods: cfDNA was extracted from the serum of 91 women with multiple uterine cancer histologies and 22 post-menopausal controls without cancer. Low molecular weight (LMW) cfDNA was separated from contaminating genomic high molecular weight cfDNA using paramagnetic bead purification and its concentration was measured using fluorometric quantification. Clinicopathologic data was abstracted from the electronic medical record. The association between serum cfDNA concentration, clinicopathologic variables, and overall survival was assessed using linear regression modelling, Cox proportional hazards modelling, and the Kaplan-Meier method. Results: Median total serum cfDNA concentration for the cohort was 69.2 ng/mL (IQR 37.4, 132.3) and median LMW cfDNA concentration was 23.8 ng/mL (IQR 14.9, 44.4). There were no significant differences in total serum cfDNA concentration with any clinicopathologic variables. However, LMW cfDNA concentration was significantly higher in serum of women with cancer (25.8 ng/mL IQR 16.0, 49.6) compared to benign controls (15.5 ng/mL IQR 9.3, 25.8 ng/ mL) (p < 0.01). It is also significantly higher among women with early stage cancer than benign controls (p < 0.01). There were also significant associations between LMW cfDNA concentration and stage of cancer (p = 0.01) and histology (p = 0.02). Patients with leiomyosarcoma and carcinosarcoma had higher cfDNA concentrations than those with endometrioid cancer. Over a median follow-up of 51.9 months, 75th percentile for overall survival for women with cancer was 24.0 months. Higher LMW cfDNA concentrations is associated with lower survival among women with cancer (p < 0.01). Conclusions: Serum LMW cfDNA concentration is associated with overall survival in women with uterine cancer, and it is higher among women with uterine cancer compared to those of controls.

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Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model

Cited by 23 publications

References 56 publications

Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Low molecular weight serum cell-free DNA concentration is associated with clinicopathologic indices of poor prognosis in women with uterine cancer

Low molecular weight serum cell-free DNA concentration is associated with clinicopathologic indices of poor prognosis in women with uterine cancer

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