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Impact of Long-Term Plasma Storage on Cell-Free DNA Epigenetic Biomarker Studies.

Author(s): Shao J, Nguyen T, Li Z

Publication: Biomolecules, 2025, Vol. 15, Page

PubMed ID: 40723799 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the cfDNA yield from unmatched plasma specimens to identify potential effects of patient diagnosis (solid tumors, acute myeloid leukemia (AML) or non-cancer), patient sex, patient age, plasma storage duration (0-12 years), delays to plasma separation (0 to ≥6 years), genomic DNA contamination, and cancer clinical stage. This study retrospectively analyzed plasma specimens collected between 2006 and 2022 from 622 patients with solid tumors (bladder, breast, colorectal, kidney, lung, ovarian, prostate, or uterine), 237 patients with acute myeloid leukemia, and 211 patients without cancer (diagnosis not specified). Blood was collected in K₂EDTA tubes and processed immediately or after 36 days (storage conditions not specified). Plasma was obtained by centrifugation at 1350 g for 10 min at 4°C and stored at -80°C for up to 14 years. Plasma was recentrifuged twice at 13,500 g for 10 min at 4°C before cfDNA extraction using the QIAamp Circulating Nucleic Acid Extraction Kit and the QIAvac system. cfDNA was quantified using the Qubit dsDNA HS Assay Kit.

   Summary of Findings:

   The median cfDNA yield from plasma was 30.36 ng/mL, with higher median yields from the plasma of AML patients than patients with solid tumors (65.49 ng/mL versus 27.28 ng/mL, P<2.0x10⁻¹⁶) or non-cancer patients (65.49 versus 34.69 ng/mL, P=1.8 x 10⁻⁵) and from non-cancer patients than patients with solid tumors (34.69 versus 27.28 ng/mL, P=1.4x10⁻⁵). No effect of plasma storage duration was found on cfDNA yield in specimens from patients with solid tumors or patients without cancer, but among AML patients, cfDNA yield was higher from plasma stored ≥12 years than 0-1.9 years (P=0.003). While cfDNA yield from the plasma of patients with solid tumors or without cancer was not significantly affected by genomic DNA contamination, the cfDNA yield from the plasma of patients with AML was significantly higher when there was genomic DNA contamination (P=0.005). cfDNA yield was higher when plasma separation occurred after 3, 4 or 5 days rather than 1 day in patients with solid tumors (P=0.009, P=0.02 and P=0.02, respectively) and after 4 days compared to 1 day in AML patients (P=0.02). There was no effect of patient age on cfDNA yield. Differences in cfDNA yield based on patient sex were limited to slightly higher yields from females with solid tumors than males (P=0.05). The cfDNA yield was higher from the plasma of patients with stage IV cancer than Stage I-III (P=0.02). Univariable quantile regression analysis that considered patient age and sex, plasma storage time, gDNA contamination, delayed plasma separation, and cancer clinical stage, identified a significant effect of plasma storage duration (P=0.001) and genomic DNA contamination (P=0.02) on cfDNA yield but these effects were limited to specimens from AML patients. In multivariable quantile regression analysis, there were no significant effects on cfDNA yield in specimens from patients with solid tumors or non-cancer patients, but among AML patients, there was a significant effect at the 50th quantile of plasma storage(P=0.008) and patient age (P=0.02) and at the 25th quantile of genomic DNA contamination (P=0.04) on cfDNA yield.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified
   • Neoplastic - Leukemia

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	AML Solid tumor Non-cancer
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	<50 years 50-59 years 60-69 years 70-79 years >80 years
   Storage	Storage duration	Processing delay of 0 days Processing delay of 1 day Processing delay of 2 days Processing delay of 3 days Processing delay of 4 days Processing delay of 5 days Processing delay of ≥6 days Plasma storage for 0-1.9 years Plasma storage for 2-3.9 years Plasma storage for 4-5.9 years Plasma storage for 6-7.9 years Plasma storage for 8-9.9 years Plasma storage for 10-11.9 years Plasma storage for ≥ 12 years
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Preaquisition	Prognostic factor	Stage I Stage II Stage III Stage IV

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2. Study Purpose

   This study compared the detection of cfDNA fragments, the presence of genomic DNA contamination, sequencing library success and number of uniquely mapped reads (UMR) in cell-free DNA extracted from unmatched plasma specimens to identify potential effects of patient diagnosis (solid tumors, acute myeloid leukemia (AML) or non-cancer), patient sex, patient age, plasma storage duration (0-12 years), delays to plasma separation (0 to ≥6 years), genomic DNA contamination, and cancer clinical stage. This study retrospectively analyzed plasma specimens collected between 2006 and 2022 from 622 patients with solid tumors (bladder, breast, colorectal, kidney, lung, ovarian, prostate, or uterine), 237 patients with acute myeloid leukemia, and 211 patients without cancer (diagnosis not specified). Blood was collected in K₂EDTA tubes and processed immediately or after 36 days (storage conditions not specified). Plasma was obtained by centrifugation at 1350 g for 10 min at 4°C and stored at -80°C for up to 14 years. Plasma was recentrifuged twice at 13,500 g for 10 min at 4°C before cfDNA extraction using the QIAamp Circulating Nucleic Acid Extraction Kit and the QIAvac system. cfDNA was quantified using the Qubit dsDNA HS Assay Kit. The fragment size profile was analyzed using the Agilent High Sensitivity Assay with a Bioanalyzer. cfDNA fragments containing 5-ydroxymethylcytosine (5hmC) were captured, PCR amplified and pair-end sequenced using a NextSeq 550 or NovaSeq 600 instrument. High-quality reads were identified using Trimmomatic and mapped using bowtie2. Statistical analysis was conducted in R.

   Summary of Findings:

   Detection of cfDNA fragments in plasma from patients with solid tumors decreased with the duration of plasma storage (P=9.4x10⁻⁴), the presence of genomic DNA contamination (P=9.5x10⁻⁶), delayed plasma separation (P=0.03) and increased with patient age (P=6.6x10⁻⁵) or cancer stage III or IV versus I (P=0.01 and P=0.002, respectively). An effect of patient age on the detection of cfDNA fragments was also observed in the plasma of non-cancer patients (P<0.05). However, in multivariate regression analysis, only genomic DNA contamination was found to be an independent factor for the detection of cfDNA fragments in solid tumors (P=0.01).

   In multivariable logistic regression, genomic DNA contamination was associated with plasma storage duration in plasma from patients with solid tumors (P=4.9x10⁻⁴) and AML (P=0.05), with delayed separation of plasma in blood from patients with solid tumors (P=0.004), male patients, in plasma from patients with solid tumors (P=0.007) and non-cancer patients (P=0.05), and those of a younger age with solid tumors (P= 0.007). Sequencing library preparation success was correlated with a high cfDNA yield in plasma from patients with AML (P =4.1x10⁻⁴), solid tumors (P=0.004) and without cancer (P=0.02); and with no genomic DNA contamination and female sex in specimens from patients with solid tumors (P=0.008 and P=8.6x10^-5, respectively).

   The number of uniquely mapped reads (UMRs) was lower in plasma from patients with solid tumors that was stored for 5-5.9 years compared to < 1 year (P =1.6x10⁻⁴) and in plasma from patients without cancer stored for 12 years compared to that stored for < 2 years (P=0.006), but was unaffected by delayed plasma separation. The number of UMRs was also higher from plasma without genomic DNA contamination, regardless of diagnosis (P=0.02 for non-cancer, P=1.4x10^-8 for solid tumors and P=0.009 for AML). The number of UMRs was not affected by patient age, but the number of UMRs was higher in specimens from females than males with solid tumors (P=1.4x10⁻⁸).  In multivariable regression analysis, longer plasma storage durations were correlated with decreased UMRs at all quantiles in specimens from non-cancer patients (25’th quantile P=0.003, 50’th quantile P=1.5x10^-9 and 75’th quantile P=5.6x10^-10) and at the 25’th quantile in specimens from AML patients (P=0.02). Genomic DNA contamination was correlated with decreased UMRs at all quantiles in specimens from patients with solid tumors (25’th quantile P=0.008, 50’th quantile P=0.01 and 75’th quantile P=0.01), at the 50’th and 75’th quantile in specimens from AML patients (P=8.6 x10^-4 and P=0.006, respectively), and at the 75’th quantile in specimens from non-cancer patients (P=0.04). A high cfDNA yield was associated with increased UMRs at the 25’th and 50’th quantile in patients without cancer (P=0.004 and P=0.03, respectively) and at the 25’th quantile in patients with AML (P=0.01). Finally, in patients with solid tumors, female sex was associated with increased UMRs at the 25’th and 50’th quantile (P=0.01 and P=0.005, respectively), but in patients with AML, female sex was associated with decreased UMRs at the 50’th quantile (P=0.03).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Leukemia
   • Other diagnoses
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	AML Solid tumor Non-cancer
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	<50 years 50-59 years 60-69 years 70-79 years >80 years
   Preaquisition	Prognostic factor	Stage I Stage II Stage III Stage IV
   Storage	Storage duration	Processing delay of 0 days Processing delay of 1 day Processing delay of 2 days Processing delay of 3 days Processing delay of 4 days Processing delay of 5 days Processing delay of ≥6 days Plasma storage for 0-1.9 years Plasma storage for 2-3.9 years Plasma storage for 4-5.9 years Plasma storage for 6-7.9 years Plasma storage for 8-9.9 years Plasma storage for 10-11.9 years Plasma storage for ≥12 years
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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