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Page 2 of 8 Moia et al. J Cancer Metastasis Treat 2019;5:67 I http://dx.doi.org/10.20517/2394-4722.2019.020

GENERAL CONCEPT ON CIRCULATING CELL FREE DNA
Liquid biopsy consists in a simple and easy sampling of peripheral blood, that can be subjected to the
[1,2]
molecular analysis of specific genomic clues released by the tumor . Because circulating tumor cells
are very rare or absent in many types of lymphomas, liquid biopsy approaches have been focused on
[3-5]
circulating tumoral DNA (ctDNA) released by lymphoma cells into the bloodstream . However, the
ctDNA represents only a fraction of the total amount of cell free DNA (cfDNA), that is derived also from
healthy cells. The cfDNA circulates in plasma at a low concentration as double-stranded DNA fragments
[6]
with a dimension < 200 base pairs . In individuals without malignancies, plasma cfDNA derives mainly
from the apoptosis of normal hematopoietic cells. cfDNA levels may also rise in para-physiological
conditions, such as trauma, burns or high exercise. In healthy subjects, the cfDNA concentration ranges
between 1 and 10 ng/mL and can reach 30ng/mL in lymphoma patients .
[7]

CFDNA ISOLATION
It is possible to selectively isolate cfDNA from plasma in order to proceed with subsequent molecular
analysis . To isolate cfDNA, a few very important technical precautions need to be observed in order
[8]
[9]
to avoid white blood cell lysis that may contaminate the cfDNA with genomic DNA (gDNA) . If using
standard collecting tubes containing ethylenediaminetetraacetic-acid (EDTA) as anticoagulant, plasma
[9]
extraction is recommended within 3 h from the collection of peripheral blood . EDTA tubes, however, are
not able to preserve the integrity of cfDNA and are not able to prevent the lysis of nucleated cells for more
than 3 h. Therefore, in order to allow the shipment of biological material, specific tubes, namely cfDNA
BCT Streck tubes (Streck), can stabilize cfDNA and can prevent the contamination by gDNA from white
[10]
blood cells for up to 14 days at a temperature between 6 and 37 °C . After collection, plasma is separated
from the corpuscular blood by centrifugation of blood samples at 800 rpm at 4 °C for 10 min. A second
centrifugation at 13,000 rpm at 4 °C for 10 min allows to pellet and remove any remaining cells. Plasma
samples are then stored in 1 mL aliquots at -80 °C until cfDNA extraction .
[8]
cfDNA can be selectively extracted using two different methods. The first method, named QIAamp
[11]
Circulating Nucleic Acid (Qiagen), relies on the use of ion-exchange resins that bind the plasma cfDNA .
The second approach relies on an automatic method that utilizes the Maxwell RSC Instrument coupled
with Maxwell RSC ccfDNA Plasma Kit (Promega). This method consists in an automated nucleic acid
purification platform that processes up to 16 samples simultaneously and allows to obtain high quality
[12]
cfDNA starting from 1 mL of plasma . After extraction, cfDNA is usually quantified by a fluorometric
assay. Approximately 30-40 ng of cfDNA are needed for subsequent molecular analysis .
[8]

CAPP-SEQ ANALYSIS AS FOR DEFINING THE LYMPHOMA GENOTYPE ON CFDNA
Since lymphoid malignancies harbor a unique molecular marker, namely the immunoglobulin (Ig)
gene rearrangement, the identification of presence of this biomarker in the cfDNA may be used to track
minimal residual disease (MRD) during the course of treatment. Once the Ig gene rearrangement has been
identified on the lymph node biopsy, it is possible to evaluate the amount of this rearrangement in the
cfDNA using Next-Generation-Sequencing (NGS) or polymerase chain reaction based methods [13,14] . Pivotal
studies have shown that the proportion of cfDNA carrying the lymphoma-specific Ig rearrangement
decreases rapidly in patients who respond to therapy and tends to remain negative in those who maintain
the response [13,14] . Conversely, the Ig rearrangement remains high in patients who do not respond to
treatment [13,14] . As expected, these patients experience both a worse progression-free survival and a worse
overall survival compared to patients with undetectable Ig gene rearrangement [13,14] . This method is very
effective in monitoring MRD but has some pitfalls. First, this method does not work in a biopsy free
manner since it requires, at the time of diagnosis, the identification of the individual patient’s specific Ig