NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Cell-free DNA extraction from urine of lung cancer patients and healthy individuals: Evaluation of a simple method using sample volume up-scaling.

Author(s): Ruppert T, Roth A, Kollmeier J, Mairinger T, Frost N

Publication: J Clin Lab Anal, 2023, Vol. , Page e24984

PubMed ID: 37991151 PubMed Review Paper? No

Purpose of Paper

This paper compared the stability of endogenous and spike-in cell-free DNA (cfDNA) in case-matched unpreserved urine and urine preserved with EDTA and/or by adjusting the pH to 8.0 with NaOH; cfDNA recovery using the standard manufacturer protocol and an in-house modification of the QIAamp MinElute Kit protocol was also compared. The authors also investigated the effects of specimen volume on cfDNA copies and compared cfDNA copies between specimens collected from males and females and between specimens collected from lung cancer patients and healthy controls.

Conclusion of Paper

The concentration of DNA (spike-in and endogenous) declined when unpreserved urine was stored at room temperature. The addition of EDTA or increasing the pH to 8.0 partially attenuated the storage-related degradation in some specimens, but the addition of both EDTA and NaOH was necessary to fully stabilize both endogenous and spike-in DNA for 10 days at room temperature.  DNA recovery from 5-100 mL of urine was comparable when extraction was with the MinElute Kit (manufacturer’s protocol) or included an in-house modification for larger volumes. DNA yield was very strongly correlated with urine volume when DNA extraction included the in-house modification (r2=0.96, P<0.0001). The copies per mL of cfDNA was significantly higher in the 19 specimens collected from females than the 13 specimens collected from men (unspecified mix of specimens from patients with cancer and volunteers) but did not differ between specimens collected from 19 cancer patients and 13 healthy volunteers. Importantly, the copies of relevant mutation sites per mL of urine were strongly correlated among genes. Finally, the authors determined that increasing the input specimen volume to > 5 mL was necessary for mutations with an allele frequency of <1%.

Studies

  1. Study Purpose

    This study compared the stability of endogenous and spike-in cell-free DNA (cfDNA) in matched unpreserved urine and urine preserved with EDTA and/or by adjusting the pH to 8.0 with NaOH; cfDNA recovery using the standard manufacturer protocol and an in-house modification of the QIAamp MinElute Kit protocol was also compared. The authors also investigated the effects of specimen volume on cfDNA copies and compared the number of cfDNA copies between specimens collected from males and females and lung cancer patients and healthy controls. Mid-stream urine was collected from thirteen healthy volunteers and nineteen lung cancer patients and centrifuged at 1200 × g for 10 min, followed 4500 × g for 10 min. All specimens were spiked with a 127 bp fragment of IS6110 M. tuberculosis. To evaluate the effects of EDTA on pH and DNA stability during storage, aliquots of urine from three volunteers were left unpreserved or preserved with EDTA (0.5 M pH 8) to reach a concentration of 40 mM, NaOH (to achieve a pH of 8.0), or EDTA and NaOH to achieve 40 mM EDTA and a pH of 8.0 and then frozen at -20°C after storage at room temperature for 0, 1, 2, 3, 5, 7 and 10 days. To test the effects of specimen volume, urine from thirteen healthy volunteers and 19 lung cancer patients was supplemented with EDTA and NaOH to achieve a concentration of 40 mM EDTA and a pH of 8.0, centrifuged, and aliquoted to create a 0.6 mL sample and a larger sample (5-100 mL). DNA was extracted from urine using the QIAamp MinElute Virus Vacuum Kit alone or with a modification that used guanidium thiocyanate and propanol for the larger specimen volumes. DNA was quantified by real-time PCR amplification of 119 bp fragments of EGFR exon 21, a 64 b, and 91 bp; a 423 bp fragment of EGFR exon 20; a 95 bp fragment of KRAS exon 2; and a 127 bp spike-in control (IS6110 M. tuberculosis).

    Summary of Findings:

    In all three specimens, the concentration of DNA (spike-in and endogenous) declined with storage of unpreserved urine at room temperature for 1-3 days. The addition of EDTA stabilized the DNA in the urine specimen with an initial pH of 8.0, but in urine specimens with an initial pH of 5.5 (6.0 after EDTA) and 5.0 (6.0 after EDTA) DNA levels decreased with room temperature storage. Similarly, addition of NaOH partially attenuated storage-related degradation of the spike-in DNA in two of the three specimens (an initial pH of 5 and 8.0) but not the remaining specimen; the storage-mediated decrease in the 119 bp fragment of EGFR was only attenuated in one of three specimens. The addition of both EDTA and NaOH stabilized endogenous and spike-in DNA in urine specimens collected from all three volunteers for at least 10 days at room temperature.

    Recovery of the 127 bp and 64 bp fragments of spike-in DNA was comparable from urine when extraction was with the MinElute Kit per the manufacturer’s instructions and with the in-house modification for the larger volume (93% versus 85% and 47% versus 55%, respectively). Similarly, the number of copies of the endogenous 119 bp fragment of DNA per mL of urine were comparable between methods (3534 and 3111, respectively). Importantly, the DNA yield was very strongly correlated with urine volume when the in-house modification was included in the extraction protocol (r2=0.96, P<0.0001). The copies of cfDNA per mL of urine were significantly higher in the 19 specimens collected from females compared to the 13 specimens collected from men (unspecified mix from patients with cancer and volunteers) (P=0.017) but did not differ between urine specimens collected from 19 cancer patients and 13 healthy volunteers. Importantly, the copies of relevant mutation sites per mL of urine were strongly correlated (r2=0.87). Finally, the authors determined that increasing the volume to > 5 mL is necessary for mutations with an allele frequency of <1%.

    Biospecimens
    Preservative Types
    • None (Fresh)
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Healthy
    Lung cancer
    Preaquisition Patient gender Female
    Male
    Biospecimen Aliquots and Components Aliquot size/volume 0.6 mL
    5-100 mL
    Biospecimen Aliquots and Components pH 5
    5.5
    8
    6
    6.5
    Real-time qPCR Specific Length of gene fragment 127 bp (spike in)
    119 bp EGFR exon 21
    64 bp EGFR exon 20
    91 bp EGFR exon 20
    423 bp EGFR exon 20
    95 bp fragment of KRAS exon 2
    Biospecimen Preservation Type of fixation/preservation EDTA
    None (fresh)
    Analyte Extraction and Purification Analyte isolation method QIAamp miniElute kit
    In house modification of QIAamp miniElute kit
    Real-time qPCR Specific Targeted nucleic acid EGFR exon 21
    EGFR exon 20
    KRAS exon 2
    Spike-in control (IS6110 M. tuberculosis)
    Storage Time at room temperature 0 days
    1 days
    2 days
    3 days
    5 days
    7 days
    10 days

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