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Page 6 of 25 Lue et al. J Cancer Metastasis Treat 2022;8:11 https://dx.doi.org/10.20517/2394-4722.2021.193

for the treatment of DHITsig DLBCLs. In an attempt to create a more applicable tool for routine use, the
authors condensed the 104-gene panel into 30 genes of essential importance, which was then added to the
Lymph2Cx assay, and referred to as DLBCL90. The DLBCL90 was able to distinguish and prognosticate
between DHITsig positive GCB-DLBCLs, DHITsig negative GCB-DLBCLs, ABC-DLBCLs and
unclassifiable tumors. The DHITsig provides further support for the adaptation of complex genetic
evaluation to help identify true poor-risk patients.

To further elaborate upon the DHITsig and classification of GCB-DLBCL, Song and colleagues combined
results obtained from IHC, GEP, DLBCL90, FISH analysis for DHL/THL, copy number analysis, and
targeted deep sequencing of 334 genes, and established four distinct groups within the broad GCB category:
GCB1 defined as DHITsig positive with TP53 inactivation; GCB2 characterized by DHITsig positivity;
GCB3 being DHITsig negative and EZB mutated and/or BCL2 translocation (EZB-like); and lastly, GCB4
which were both negative for DHITsig and EZB-like characteristics . Using 87 primary patient samples,
[6]
patients assigned to the GCB2 and GCB4 categories displayed superior prognosis after R-CHOP therapy
compared to the GCB1 and GCB3. This survival advantage was validated in an additional 188 GCB-DLBCL
cases. Along these lines, the majority of cases fell under the GCB4 group (51%), perhaps, driving our older
observation that GCB-DLBCLs, as a whole, have favorable outcomes after standard R-CHOP. However, it is
clear from this study and others that not all GCB-DLBCLs (and ABC-DLBCLs) behave the same,
supporting the need to validate a universal assay that can quickly output these novel molecular sub-
classification designations.

Limitations of novel genetic classifications
Although these novel classification systems and assays add to our understanding of DLBCL and have
prognostic implications, the question still persists on how applicable and readily available these assays are to
everyday use. We have been able to apply some of these diagnostics to clinical trial studies, however, we
have yet to evaluate these new hierarchies in a prospective manner. Ultimately, the true implication of these
novel clusters or sub-classifications requires prospective validation in parallel with the evaluation of novel
therapeutics applied in a precision medicine fashion. Moreover, these assays often rely upon central
laboratories, which illustrates another limitation - the ability to replicate these diagnostic tests from one
laboratory to another with ease and efficiency, all the while maintaining the accuracy of the assay. Until we
are able to quickly, and without compromising accuracy, replicate these assays in local or commercialized
laboratories, the pervasive use of these diagnostic tools will be limited to clinical trial and academic
applications. Furthermore, as we continue to probe into the genetic background of DLBCL, we are likely to
find even smaller subsets based on unique genetic and mutational characteristics, which in turn will impact
clinical trial design. Although these classifications are not yet ready for universal use, the wealth of
information obtained from these analyses has revealed potential targets for novel drug development and will
greatly assist in our attempts to improve the clinical outcomes of DLBCL patients.

Potential of circulating tumor DNA in the management of Diffuse Large B-cell Lymphoma
Liquid tumor biopsies have been developed in both solid and hematological malignancies as both a
predictive and diagnostic tool that obviates the need for potentially invasive biopsies, and is currently an
emerging tool for the DLBCL management. Circulating cell-free DNA (cfDNA) are DNA fragments that are
routinely released into circulation by apoptotic or necrotic cells. In general, cfDNA are cleared from the
[30]
plasma by the renal system, liver and/or spleen as well as nucleases . Circulating tumor DNA (ctDNA)
specifically refers to cfDNA fragments that originate from tumor cells and thus far has been investigated in
diagnostic, therapeutic and prognostic scenarios. Polymerase chain reaction (PCR)-based methods that
identify single point mutations, such as MYD88 L265P in primary central nervous system lymphoma [31,32] ,
were one of the earliest iterations by which ctDNA was evaluated in lymphoma patients. This method is