Impact of cell-debris and room-temperature storage on urine circulating tumor DNA from hepatocellular carcinoma.
Author(s): Kim AK, Lin SY, Wang Z, Luu H, Hamilton JP, Wei Song, Su YH
Publication: J Mol Diagn, 2023, Vol. , Page
PubMed ID: 37813297 PubMed Review Paper? No
Purpose of Paper
This paper investigated whether fetal DNA (Y-chromosome) and hepatocellular carcinoma (HCC) DNA markers could be detected in urinary cellular debris and compared the DNA size distribution, DNA levels, and detection of HCC mutations in matched urine aliquots that were immediately frozen with those stored at room temperature for seven days before freezing.
Conclusion of Paper
The fetal Y chromosome was detected in the cellular debris of all six specimens evaluated. Two HCC markers, mutated TERT and methylated RASSF1A (mRASSF1A), were found at higher levels in the debris pellet than in the supernatant. The authors concluded that transrenal DNA, including HCC DNA, can be found in cellular debris. For 20 of the 21 pairs of urine specimens evaluated, the aliquot stored at room temperature for 7 days had a similar size distribution pattern as the urine aliquot that was immediately frozen. Levels of TP53, CTNNB1, and TERT as well as detection of the HCC mutation were unaffected when urine was stored at room temperature for 7 days.
Studies
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Study Purpose
This study investigated whether fetal DNA (Y-chromosome) and HCC DNA markers could be detected in urinary cellular debris. A total of six archival urine specimens collected from two women who were pregnant with a male fetus (5 specimens from one patient, 1 specimen from a second patient) were spiked with SPKN DNA; additional details pertaining to specimen collection and storage were not provided. Additionally, urine specimens collected from 5 patients diagnosed with HCC were retrieved from the archive (storage conditions not specified). Urine was centrifuged at 1,500 RPM at 4°C for 10 mins to separate the supernatant and cellular debris. DNA was isolated from uncentrifuged urine, the supernatant, and cellular debris using the JBS Urine Total DNA Isolation Kit. The fragment size profile of the isolated DNA was characterized using Agilent’s High Sensitivity D5000 Screentape Assay on a TapeStation. DNA was quantified by ddPCR using a TERT PCR Quantification Kit, a Y103 Chromosome quantification assay and methylation was measured with RASSF1a and mGSTP1 assays.
Summary of Findings:
Both the spike-in control DNA and fetal Y chromosomal DNA were detected in supernatant of maternal urine specimens, with 47.8-91.1% of the spike-in recovered in the supernatant. As expected, the spike-in control was barely detected in the cellular debris isolated from maternal urine specimens, whereas the fetal Y chromosome was detected in the cellular debris of all six maternal urine specimens. DNA present in the pelleted debris of urine collected from HCC patients was less than 1 kb; urine specimens of three HCC patients contained clear mononucleosomal-sized peaks. Importantly, the HCC markers mutated TERT-124 and mRASSF1A were found at higher levels in the urine debris pellet than in the supernatant. The authors concluded that transrenal DNA, including HCC DNA, is detectable in the cellular debris of urine specimens.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Pregnant
Platform:
Analyte Technology Platform DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Biospecimen components Supernatant
Cell debris
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Study Purpose
This study compared the DNA size distribution, DNA levels, and detection of HCC mutations in matched urine aliquots that were stored at room temperature for seven days before freezing with those that were immediately frozen. Urine was collected from 21 patients diagnosed with hepatocellular carcinoma (HCC) and matched aliquots were placed in EDTA tubes and frozen immediately at -20°C or stored for 7 days at room temperature before freezing -20°C. DNA was extracted using the JBS Urine Total DNA Isolation Kit. The fragment size profile of isolated DNA was characterized using Agilent’s High Sensitivity D5000 Screentape Assay on a TapeStation. DNA was quantified by ddPCR using assays for TP53, CTNNB1, and TERT and methylation was quantified using assays for RASSF1a and mGSTP1.
Summary of Findings:
Eight of the 21 urine specimens evaluated had distinct nucleosomal DNA peaks, whereas the remaining specimens had a broader DNA distribution. For 20 of the 21 pairs of urine specimens evaluated, the aliquot stored at room temperature for 7 days had a similar size distribution pattern as the urine aliquot that was immediately frozen. Levels of TP53, CTNNB1, and TERT were comparable between the urine specimens that were stored for 7 days at room temperature prior to freezing and those that were frozen immediately. Finally, 18 of 21 urine specimens had a detectable HCC mutation; the HCC mutation detection rate did not differ between urine aliquots that were stored and those that were immediately frozen.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 0 days
7 days