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Plasma or Serum: Which Is Preferable for Mutation Detection in Liquid Biopsy?

Author(s): Pittella-Silva F, Chin YM, Chan HT, Nagayama S, Miyauchi E, Low SK, Nakamura Y

Publication: Clin Chem, 2020, Vol. , Page

PubMed ID: 32516802 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared cfDNA yield, size distribution, NGS metrics, and mutation detection in matched plasma and serum specimens. Mutations detected in plasma and serum were compared to those in tissue specimens obtained at diagnosis. Blood was collected simultaneously from five healthy, 20 lung cancer, and 28 colorectal cancer patients into Na₂EDTA and plain tubes. Matched tissue biopsy specimens were collected at diagnosis but no details of tissue processing were provided. Blood was processed to serum after a 30 min clot time and to plasma within 2 h of collection. For some experiments, plasma and serum from a healthy individual were spiked with DNA carrying clinically relevant mutations. Serum and plasma were obtained by centrifugation at 2000 x g for 10 min followed by 16000 x g for 10 min and frozen at -80°C. cfDNA was extracted from plasma and serum using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit and quantified using the Qubit dsDNA HS Assay Kit. DNA fragment size was evaluated using High-Sensitivity D5000 ScreenTape Kit on the TapeStation system. NGS libraries were constructed from cfDNA from healthy individuals and patients with colorectal carcinoma using Oncomine Pan-Cancer Cell-Free Assay and from patients with lung cancer using the Oncomine Lung cfDNA Assay libraries. Libraries were sequenced using the Ion S5.

   Summary of Findings:

   The cfDNA yield was higher in plasma from patients with lung cancer or colorectal carcinoma than healthy controls (42.2%, P=0.022 and 28.4% P=0.23, respectively) but not in serum. The DNA yield was higher from serum than plasma of healthy patients (36.7%, P=0.029) and those with lung cancer (54.8%, P=0.044) but not in patients with colorectal cancer. Although the majority of the DNA was 10-250 bp, serum also showed peaks greater than 500 bp. Serum had a higher percentage of fragments >500 bp than plasma in healthy individuals (14% versus 9%, P=0.004) and in patients with lung (32% versus 15%, P=0.0001) or colorectal cancer (16% versus 13%P=0.015). Serum and plasma had comparable molecular tagging efficiency, library concentration, percentage of reads mapped, and median depth but median absolute pairwise difference (MAPD) was higher in serum than plasma for healthy patients (P=0.005) and for patients with colorectal carcinoma (P=0.002) or lung cancer (P=0.004). The mean molecular depth was correlated with input in plasma and serum from patients with colorectal carcinoma (r=0.9283, P<0.0001 and r=0.8771, P<0.0001; respectively) and patients with lung cancer (r=0.9157, P<0.0001 and r=0.9064, P<0.0001; respectively).

   DNA concentrations were 1.4-fold higher in serum than plasma resulting in approximately 50% lower calculated allele frequency and lower absolute counts for spiked-in mutations in serum than plasma (P=0.004 and P=0.002, respectively). Using specimens from lung cancer patients, genetic alterations were found in both the plasma and serum specimen of half the patients, in just the serum in one patient, and in just the plasma of one patient. However, when broken down by mutation, only eight SNVs and one CNV were found in both plasma and serum of the same patients, nine SNVs with low AF were found only in plasma, and five SNVs in tumor protein 53 (TP53) were only found in serum. Importantly, the MAF was higher in plasma than serum for each of the mutations and was significantly higher in plasma than serum specimens (P=0.042). When compared to matched tumor specimens, three of seven had the same mutation detected in cfDNA from plasma and serum as the tumor and two had novel mutations identified in the cfDNA; one of which was only found in the plasma. In specimens from colorectal carcinoma patients, mutations were detected in both the plasma and serum specimens of 60.7% of patients and in just the plasma of an addition 25%. Of the 58 mutations identified, 26 were found in both samples from the same patients, another 26 were only found in the plasma specimens and six were only detected in serum specimens. Similar to lung cancer, the AFs were a median of 1.9-fold higher in plasma than serum (P=0.001) but the median molecular sequencing depth was comparable in serum and plasma. Of 11 patients with mutations identified in tumor, the same mutation was detected in both the plasma and serum for two cases and in plasma for only three more, but AF was higher in plasma than serum in all cases.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Lung cancer Colorectal carcinoma
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Biospecimen Acquisition	Biospecimen location	Colorectal biopsy Lung biopsy Blood
   Next generation sequencing Specific	Template/input amount	5-20 ng

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