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rearrangement on the lymph node tissue biopsy. Once the patient’s unique Ig rearrangement has been
identified on the tissue biopsy, it is then possible to track it on the liquid biopsy to evaluate MRD. Second,
[15]
this method does not allow a comprehensive disease genotyping on the liquid biopsy .
To overcome these limits, the use of Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) has
[16]
been established as a reliable tool to genotype cancer patients . This molecular strategy utilizes disease
specific probes that selectively capture a set of exonic and intronic regions known to be recurrently affected
in a particular cancer type. The selected targets are then amplified and sequenced by NGS allowing the
detection of the specific tumor mutational profile of a patient. This method, linked to a stringent bio-
informatic analysis, allows to detect mutations in cfDNA with a high sensitivity, identifying mutations
with allelic frequency as low as 0.02%. In addition, CAPP-Seq allows the identification of single nucleotide
variants, as well as insertion/deletions, copy number alterations and rearrangements, thus covering a large
[16]
variety of genomic alterations of human cancers .

APPLICATION OF LIQUID BIOPSY IN LYMPHOMA
Lymph node biopsy is the gold standard for the diagnosis of lymphoma, but genetic lesions identified in the
tissue biopsy may not reflect the entire molecular complexity of every single patient with lymphoma [17,18] .
Consistently, in an individual patient, lymph nodes at different anatomical sites, as well as different areas
[19]
of the same lymph node, may show different genetic profiles . However, outside of clinical studies aimed
at investigating the clonal heterogeneity of lymphomas, multiple biopsies are not routinely performed
in lymphoma patients for both practical and ethical concerns. On these grounds, once a diagnosis of
lymphoma is performed on a tissue biopsy, liquid biopsy may be used to explore the entire mutational
landscape of lymphoma, since this approach has the potential to collect the tumor cfDNA deriving from
[15]
most, or potentially all, of the different sites of tumor involvement in the body [Figure 1] . In particular,
a liquid biopsy CAPP-Seq approach may be used to: (1) genotype lymphoma patients; and to (2) evaluate
treatment response [Figure 2].

LIQUID BIOPSY AS A TOOL FOR GENOTYPING LYMPHOMA PATIENTS
In DLBCL, liquid biopsy allows to identify at least one somatic non-synonymous mutation per patient in
over 70% of cases [20,21] . The mutational profile identified by liquid biopsy reflects that revealed by DLBCL
[22]
genomic studies performed on the lymph node biopsies . More precisely, the most common genetic
lesions found by liquid biopsy reflect those detected on the tissue biopsy, and comprise alterations of
KMT2D, followed by TP53, CREBBP, and PIM1. In addition, EZH2 and BCL2 mutations, as expected, are
more frequently found in germinal center (GC) DLBCL, whereas TNFAIP3 and PIM1 mutations are more
[20]
frequent in non-GC DLBCL . In addition, by comparing the mutations identified in cfDNA with those
identified in gDNA extracted from the tissue biopsy, cfDNA appears to be representative of most of the
mutations that occur in > 20% of the alleles of the tumor biopsy, with a sensitivity > 90% and a specificity
of ∼100%. Furthermore, a fraction of mutations has been found exclusively in cfDNA, conceivably because,
due to spatial tumor heterogeneity, they are restricted to clones that are anatomically distant from the
biopsy site .
[20]
Regarding cHL, the rarity of neoplastic Hodgkin and Reed-Sternberg (HRS) cells in the biopsy has
[23]
limited the assessments of the genetic landscape of the disease . In this context, liquid biopsy may be a
reliable tool to overcome this limit. Analogous to DLBCL, it has been demonstrated that cfDNA analysis
in cHL mirrors the genetics of HRS cells micro-dissected from the tissue biopsy. Consistently, most of
cHL mutations are identified in the tissue biopsy as well as in the cfDNA and some more mutations
[23]
are identified only in cfDNA, conceivably because they are derived from different anatomical sites .
Genotyping of cHL on the liquid biopsy has allowed to refine the current knowledge of cHL genetics.