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relatively low cost and efficient but has the limitation of analyzing only select mutations with only a few
mutations occurring with a high enough frequency to make PCR broadly applicable to disease subtypes.

As such, next-generation-sequencing (NSG) has revolutionized our ability to evaluate a panel of relevant
mutations in a lymphoma-subtype or cancer-specific way. In order to achieve a broader applicability in
lymphoma, the detection of clonotypic immunoglobulin (Ig) rearrangements through high throughput
sequencing (IgHTS) was developed and used in early minimal residual disease (MRD) and relapse detection
studies [33,34] . However, IgHTS is limited by the amount of total cfDNA present in plasma and cannot provide
information regarding mutational profiling or genetic drivers. Unlike IgHTS, the cancer personalized
profiling by deep sequencing (CAPP-seq) is able to target many genetic aberrations with a sensitivity of 2.5
[35]
5
part per 10 cfDNA and is able to be diagnostic and therapeutic by providing mutational profiling. In fact,
as compared to IgHTS, CAPP-seq has a higher sensitivity in both tumor and plasma ctDNA detection, and
is able to provide COO information, as well as detect DLBCL relapse earlier than IgHTS .
[36]
Several studies have evaluated baseline ctDNA and correlated levels to overall tumor burden [33,34] , as well as
clinical outcomes with patients harboring > 2.5 log he/mL of ctDNA at baseline having inferior event free
survival (EFS) at 24 months in both the front-line and salvage settings . Moreover, the role of interim
[37]
ctDNA monitoring in the upfront and salvage setting revealed two excellent responder groups: the early
molecular response (EMR: 2 log decrease after 1 cycle of treatment) and major molecular response (MMR:
2.5 log decrease after 2 cycles of therapy) . Treatment naive patients who achieved an EMR or MMR had
[37]
superior outcomes as measured by EFS and OS at 24 months compared to patients who did not achieve
these landmarks (EMR P = 0.0015, and P < 0.001; MMR, P < 0.001 and P = 0.047, respectively). In the
patients receiving salvage therapy, EMR was also prognostic and associated with improved EFS (EFS 100%
vs. 13%, P = 0.011).

In order to improve the depth and sensitivity for ctDNA MRD, Kurtz and colleagues developed phased
variant enrichment and detection by sequencing (PhasED-seq) which identifies multiple clustered
mutations on a single DNA molecule. These mutations are present on the same strand of DNA and occur in
predicted areas of the genome, driven by aberrant somatic hypermutation by activation-induced
deaminase . In fact, PhasED-seq was able to outperform CAPP-seq in the ability to detect MRD after 2
[38]
cycles of therapy as well as at end of treatment.

Ultimately, ctDNA is a promising and emerging biomarker in lymphoma, including DLBCL, that can
overcome potential limitations of tissue biopsies and radiographic scans all the while providing
molecular/tumor profiling information, risk stratification and disease/surveillance monitoring. The bar for
sensitivity of ctDNA detection continues to rise, with the newest method of PhasED-seq improving the
depth of detection compared to prior NSG methods. Although ctDNA seems logistically an appropriate
biomarker and diagnostic tool to further develop, it inherently does not provide information regarding the
interaction of the tumor and tumor microenvironment and has its limitations with low variant allele
frequencies. Ultimately, prospective studies are needed to validate the exact role of ctDNA for MRD
assessment, the development of possible risk-adapted trials (de-escalation or escalation of treatment), and
lastly, as clinical surveillance following the completion of treatment.

EVOLVING TREATMENT OPTIONS
Standard of care
Despite extensive work that has been accomplished in understanding and elucidating the diverse
pathological drivers of DLBCL, several attempts at improving our first-line therapy for DLBCL have failed.