Circulating cell-free DNA from plasma undergoes less fragmentation during bisulfite treatment than genomic DNA due to low molecular weight

Abstract Background: The objective of this study was to evaluate the association of serum cell-free DNA (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 = 2.4 x 10-4). It is also significantly higher among women with early stage cancer than benign controls (p = 0.002). There were also significant associations between LMW cfDNA concentration and stage of cancer (p = 0.01) and histology (p = 0.016). 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 = 4.6 x 10-4).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.

Download Full-text

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

10.21203/rs.3.rs-22988/v2 ◽

2020 ◽

Author(s):

Gregory M. Gressel ◽

Elaine C. Maggi ◽

Bryan E. Harmon ◽

Kenny Q. Ye ◽

D.Y.S. Kuo ◽

...

Keyword(s):

Molecular Weight ◽

Overall Survival ◽

Poor Prognosis ◽

Early Stage ◽

Uterine Cancer ◽

Cox Proportional Hazards ◽

Total Serum ◽

Low Molecular Weight ◽

Cell Free Dna ◽

Free Dna

Abstract 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.

Download Full-text

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

Journal of Translational Medicine ◽

10.1186/s12967-020-02493-8 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Gregory M. Gressel ◽

Elaine C. Maggi ◽

Bryan E. Harmon ◽

Kenny Q. Ye ◽

D. Y. S. Kuo ◽

...

Keyword(s):

Molecular Weight ◽

Poor Prognosis ◽

Uterine Cancer ◽

Low Molecular Weight ◽

Cell Free Dna ◽

Dna Concentration ◽

Free Dna

Download Full-text

Baseline mutation profiling of circulating cell-free DNA from healthy individuals to improve the detection accuracy of circulating tumor DNA in cancers.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23057 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23057-e23057

Author(s):

Geng Tian ◽

Ligang Xia ◽

Zhoufang Li ◽

Xiaohua Li ◽

Feiyue Xu ◽

...

Keyword(s):

Genomic Dna ◽

Circulating Tumor Dna ◽

Clinical Diagnostics ◽

Detection Accuracy ◽

Healthy Individuals ◽

Cell Free Dna ◽

Free Dna ◽

Tumor Dna ◽

Mutation Profiling ◽

Circulating Cell Free Dna

e23057 Background: Cell-free circulating tumor DNA (ctDNA) has emerged as an effective blood-based biomarker for clinical diagnostics in cancers, improving the accuracy and efficiency of cancer treatment for clinic. However, the somatic mutations originated from inheritance or normal biological metabolism in circulating cell-free DNA (cfDNA) in plasma can largely interfere with the detection of ctDNA. The baseline for ctDNA evaluation is urgently needed for accuracy identification of ctDNAs in patients. Methods: Averagely 20 ml blood samples were collected from more than 1200 individuals including healthy volunteers, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer patients, etc. Genomic DNA from white blood cells and cfDNA from plasma were extracted and constructed as sequencing libraries using a panel contains 50 cancer-associated genes, respectively for each sample. Then they are subjected to ultra-deep sequencing with average depth > 40000 folds covering ~21K nucleotide regions. Results: The background somatic mutation frequencies were detected in genomic DNA and cfDNA in healthy controls. The results showed that most of mutations in cfDNA were consistent with those in genomic DNA. Using data from 1200 individuals, we generated the baseline mutation profiling of cfDNA, which was referred in the ctDNA determination in cancer samples, which significantly improved the accuracy of ctDNA detection compared with tissue biopsy. Conclusions: Our studies demonstrated the importance of sequencing both cfDNA and genomic DNA for ctDNA detection in cancers. We also determined the baseline mutation profiling of circulating cfDNA from more than 1200 healthy individuals and confirmed the value of it by comparing with DNA sequencing data in cancer tissue. Our work offers clue on how to improve the detection accuracy of circulating tumor DNA in early cancer diagnosis.

Download Full-text

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

10.1101/189340 ◽

2017 ◽

Cited By ~ 1

Author(s):

Geng Tian ◽

Xiaohua Li ◽

Yuancai Xie ◽

Feiyue Xu ◽

Dan Yu ◽

...

Keyword(s):

Lung Cancer ◽

Genomic Dna ◽

Tumor Tissue ◽

Circulating Tumor Dna ◽

Driver Genes ◽

Cell Free Dna ◽

Lung Cancer Patients ◽

Free Dna ◽

Tumor Dna ◽

Circulating Cell Free Dna

BackgroundRemarkable 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.MethodsWe 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.ResultsDetection 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.ConclusionWe 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.

Download Full-text