An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens: sample preparation for epigenetic analysis

Hasinger, Oliver; König, Thomas; Schlegel, Anne; Weiß, Günter

doi:10.1186/s13104-019-4595-3

Cited by 6 publications

(4 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The choice of follow-up strategy will depend on local resources available and availability of well trained cytotechnicians. Automated workflows for methylation analysis are currently being developed, which combined with robust and user friendly assays can generate results within a day, thereby also impacting costs [54,55].…”

Section: Discussionmentioning

confidence: 99%

Posttreatment monitoring by ASCL1/LHX8 methylation analysis in women with HIV treated for cervical intraepithelial neoplasia grade 2/3

Vink

Kremer

Lissenberg‐Witte

et al. 2022

Aids

View full text Add to dashboard Cite

Objective:Women with HIV (WWH) have an increased risk to develop recurrent cervical intraepithelial neoplasia grade 2/3 (rCIN2/3) after treatment compared with HIV-negative women. Therefore, appropriate posttreatment monitoring of WWH is important. This study evaluates the performance of ASCL1 and LHX8 methylation analysis as posttreatment monitoring test in WWH treated for CIN2/3, as alternative to cytology or human papillomavirus (HPV) as follow-up test.Design:Prospective observational cohort study.Methods:WWH treated for CIN2/3 by large loop excision of the transformation zone (LLETZ) (n = 61) were invited for follow-up study visits at 1, 2.5 and 4 years after baseline. Baseline and follow-up cervical scrapes were tested for cytology, HPV and DNA methylation of ASCL1 and LHX8 genes. The performance of these strategies for the detection of rCIN2/3 was evaluated in the first follow-up cervical scrape.Results:Thirteen (21.3%) rCIN2/3 lesions were detected within 4 years of follow-up. In women without rCIN2/3 in follow-up, methylation levels of ASCL1 and LHX8 decreased significantly after LLETZ treatment (P = 0.02 and 0.007, respectively). In women with rCIN2/3, methylation levels remained high after LLETZ treatment. The 4-year rCIN2/3 risk was 4.9% (95% CI: 0.6–16.5) for ASCL1/LHX8-negative women, 8.1% (95% CI: 1.7–21.9) for HPV-negative women and 7.7% (95% CI: 2.1–18.5) for cytology-negative women.Conclusion:A negative ASCL1/LHX8 methylation test in follow-up is associated with a low rCIN2/3 risk and could serve as an objective test of cure and well tolerated alternative for HPV and/or cytology screening in the posttreatment monitoring of WWH.

show abstract

Section: Discussionmentioning

confidence: 99%

Posttreatment monitoring by ASCL1/LHX8 methylation analysis in women with HIV treated for cervical intraepithelial neoplasia grade 2/3

Vink

Kremer

Lissenberg‐Witte

et al. 2022

Aids

View full text Add to dashboard Cite

show abstract

“…For the pretreatment process, target enrichment with equal volume of concentrated reagent like PEG 6000 could be applied. In addition, increasing the amounts of GT and carrier RNA might be used to ensure sufficient cfDNA output 67‐70 . The similar strategy could also be used for saliva and other type of samples.…”

Section: Discussionmentioning

confidence: 99%

Development and validation of a flexible DNA extraction (PAN) method for liquid biopsy of multiple sample types

Chen

Liu

et al. 2021

Clinical Laboratory Analysis

View full text Add to dashboard Cite

Background Liquid biopsy is gaining increasing popularity in cancer screening and diagnosis. However, there is no relatively mature DNA isolation method or commercial kit available that is compatible with different LB sample types. This study developed a PAN‐sample DNA isolation method (PAN method) for liquid biopsy samples. Methods The PAN method has two key steps, including biosample‐specific pretreatments for various LB sample types and high concentration guanidine thiocyanate buffer for lysis and denaturation procedure. Subsequently, the performance of PAN method was validated by a series of molecular analyses. Results The PAN method was used to isolate DNA from multiple sample types related to LB, including plasma, serum, saliva, nasopharyngeal swab, and stool. All purified DNA products showed good quality and high quantity. Comparison of KRAS mutation analysis using DNA purified using PAN method versus QIAamp methods showed similar efficiency. Epstein‐Barr virus DNA was detected via Q‐PCR using DNA purified from serum, plasma, nasopharyngeal swab, and saliva samples collected from nasopharyngeal carcinoma patients. Similarly, methylation sequencing of swab and saliva samples revealed good coverage of target region and high methylation of HLA‐DPB1 gene. Finally, 16S rDNA gene sequencing of saliva, swab, and stool samples successfully defines the relative abundance of microbial communities. Conclusions This study developed and validated a PAN‐sample DNA isolation method that can be used for different LB samples, which can be applied to molecular epidemiological research and other areas.

show abstract

“…Bisulfite treated DNA (bisDNA) was prepared from plasma with reagents from the commercially available Epi BiSKit (Epigenomics AG, Berlin) [ 20 ] using an automated protocol on the Tecan Evo 200 liquid handling platform (Männedorf, CH) [ 21 ]. In order to ensure that all samples from the same patient were comparable, multiple plasma samples from an individual patient were pooled and equally distributed into 3.5 ml aliquots, processed in parallel and stored at − 20 °C.…”

Section: Methodsmentioning

confidence: 99%

Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients. Methods DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms. Results The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9). Conclusions Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples. Trial Registration: Test set study—ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

show abstract

An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens: sample preparation for epigenetic analysis

Cited by 6 publications

References 7 publications

Posttreatment monitoring by ASCL1/LHX8 methylation analysis in women with HIV treated for cervical intraepithelial neoplasia grade 2/3

Posttreatment monitoring by ASCL1/LHX8 methylation analysis in women with HIV treated for cervical intraepithelial neoplasia grade 2/3

Development and validation of a flexible DNA extraction (PAN) method for liquid biopsy of multiple sample types

Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study

Contact Info

Product

Resources

About