Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Circulating Cell-Free DNA Assessment in Biofluids from Children with Neuroblastoma Demonstrates Feasibility and Potential for Minimally Invasive Molecular Diagnostics.

Author(s): Lodrini M, Wünschel J, Thole-Kliesch TM, Grimaldi M, Sprüssel A, Linke RB, Hollander JF, Tiburtius D, Künkele A, Schulte JH, Lankes E, Elgeti T, Hundsdörfer P, Astrahantseff K, Simon T, Eggert A, Deubzer HE

Publication: Cancers (Basel), 2022, Vol. 14, Page

PubMed ID: 35565208 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare cfDNA yield and gDNA contamination in urine, blood plasma, bone marrow plasma and CSF specimens obtained from patients with neuroblastoma, healthy patients, and pediatric patients without malignancies. Potential effects on cfDNA yield associated with differences in specimen volume and delayed centrifugation of blood were also investigated. The authors also investigated the use of cfDNA yield as a marker of cancer relapse and assessed the potential of blood or bone marrow plasma for the detection of copy number variations or mutations in the tumor.  Blood, bone marrow, cerebrospinal fluid and urine were collected from 84 pediatric patients (median 23.2 months old, range: 1-162 months old) with low (28 patients), intermediate (6 patients) or high (50 patients) risk neuroblastoma. Tumor specimens were also available from 3 patients (details not provided).  Blood was also collected from 25 pediatric patients (median 72.4 months old) with non-malignant conditions (diagnosis not specified) and 43 healthy bone marrow donors (median age 26.3 years, range: 4.5-46.4 years). Longitudinal blood specimens were collected from 7 patients with high-risk neuroblastoma, prior to, during and after first-line treatment; blood specimens collected after a cancer relapse were available from 4 additional patients. Blood and bone marrow specimens were collected in EDTA tubes and transported to the laboratory (median duration was 1 h for blood and 0.9 h for bone marrow). CSF and urine were collected in plain polypropylene tubes and transported to the laboratory (median duration was 1 h for CSF and 1.3 h for urine).  Upon arrival at the laboratory, blood, CSF and urine were centrifuged at 1,900 g for 7 min, while bone marrow specimens were centrifuged at 450 g for 7 min followed by 3,250 g for 10 min. Plasma, CSF, urine and bone marrow plasma were then frozen at -80°C. After thawing, specimens were centrifuged at 2,000 g for 5 min followed by 20,000 g for 5 min. cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit and concentrated with the DNA Clean and Concentrator-5 Kit. cfDNA yields and size distributions were analyzed using the cfDNA ScreenTape assay. MYCN and ALK copy numbers and the presence of ALK mutations (3522, C > A and 3824, G > A) were evaluated by droplet digital PCR (ddPCR). DNA was extracted from three tumor specimens using the Qiagen Puregene Core Kit or the QIAamp DNA Mini Kit. DNA was quantified on a Qubit Fluorometer.

   Summary of Findings:

   Plasma isolated from the 18 blood specimens that were processed after a >4 h delay to centrifugation had higher levels of cfDNA than plasma obtained from the 175 blood specimens subjected to a delay that was ≤4 h (P<0.05), an increase that was due to a higher levels of gDNA.  When blood plasma specimens from cancer and healthy patients were considered together 85.5% of blood plasma specimens contained predominantly cfDNA while just 9.1% contained predominantly gDNA. When broken down patient diagnosis, gDNA was predominant in 62.3% of bone marrow plasma specimens from patients with neuroblastoma but 84.2% of bone marrow plasma specimens from healthy patients. Interestingly, DNA size distributions did not differ when hemolytic/cloudy and non-hemolytic bone marrow plasma were compared.  The predominant DNA peak was gDNA in the 26.7% of CSF specimens with detectable DNA peaks.  In urine, a broad cfDNA DNA peak occurred in 91.7% of specimens from patients with neuroblastoma and 40% of healthy controls, while gDNA was predominant in the remaining 8.3% and 60% of patients, respectively. The authors note that differences in DNA size distribution between neuroblastoma patients and healthy patients were largest in bone marrow plasma and urine specimens. Neuroblastoma patients had cfDNA levels that were 5.2-, 16.3- and 14.2-fold higher in blood, bone marrow plasma, and urine, respectively, than healthy pediatric control patients. Although significantly lower median specimen volumes were collected from patients <18 months of age compared to older patients (P<0.01), sufficient specimen volumes were collected for the majority of patients, as less than 4.9% of blood specimens and 14.3% of bone marrow specimens had a specimen volume below 0.5 mL. The percentage of blood specimens with an insufficient amount of cfDNA (less than the 10 ng of cfDNA required for a second sequencing run) declined when the volume of specimen collected increased: 79.3%, 61.4%, 50.6% or 44.8% of specimens with a volume of 0.5, 1.0, 1.5 or 2.0 mL of blood plasma, respectively. None of the 15 CSF specimens contained > 1 ng of cfDNA. The percentage of urine specimens that yielded ≤ 1 ng cfDNA was high but declined when larger specimen larger volumes were collected (91.7%, 75%, 75% and 58.3% of when urine specimen volumes were 0.5, 1.0, 1.5 and 2.0 mL, respectively), indicating larger urine volumes are needed for cfDNA analysis.  Five of the seven patients that provided longitudinal blood specimens had cfDNA levels > 50 ng/L in the first sample collected prior to patient treatment; cfDNA levels declined after treatment in these five longitudinal specimens to stabilizing at levels comparable to those observed in the pediatric control group. Blood specimens from the remaining two patients that underwent longitudinal sampling displayed no change in cfDNA levels after treatment…Blood specimens collected from four of the seven patients upon cancer relapse did not show a clear relationship between relapse and cfDNA levels, as cfDNA levels were lower than in the initial sample in 2 patients but higher in the other 2, which led the authors to conclude that cfDNA levels are not a reliable indicator of cancer relapse.  MYCN copy number status was accurately detected using a 1.4 mL blood specimen and a 2.0 mL bone marrow specimen from the one patient examined with this CNV. ALK pR1275Q was detected in one case when no disease was detected via standard bone marrow diagnostics and in another case the mutation had not been detected in the tumor specimen despite limited volumes of blood plasma (0.85 mL and 1.3 mL, respectively) and bone marrow plasma (1.5 mL and 0.8 mL, respectively).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma
   • Bodily Fluid - Cerebrospinal Fluid
   • Bodily Fluid - Urine
   • Bodily Fluid - Bone Marrow

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Not specified
   • Neoplastic - Pediatric

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Pediatric non-neoplastic diagnosis Neuroblastoma
   Biospecimen Acquisition	Biospecimen location	Blood plasma Bone marrow plasma Urine CSF
   Biospecimen Aliquots and Components	Aliquot size/volume	0.5 mL 1.0 mL 1.5 mL 2.0 mL
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Acquisition	Time of biospecimen collection	Initial collection During treatment After treatment At relapse
   Storage	Storage conditions	<4 h >4 h
   Preaquisition	Patient age	<18 months >18 months

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