Can Plasma Track CD20? The Limits of ctDNA After Anti-CD20 Therapy

The clinical question

CD20 is one of the most consequential targets in hematologic oncology. It's the antigen that anti-CD20 monoclonal antibodies, rituximab and its successors, are built to engage, and its expression on malignant B cells is foundational to that entire therapeutic class [5]. The problem clinicians run into is that CD20 isn't static. Under the selective pressure of anti-CD20 therapy, tumors can lose CD20 expression, and that loss carries real therapeutic implications: a lymphoma that was CD20-positive at diagnosis may present as CD20-negative at relapse, changing whether continued targeting of that antigen makes biological sense [5].

Historically, the only way to know whether CD20 had been lost was to go back to tissue, a repeat biopsy scored by immunohistochemistry or flow cytometry, with expression read as present versus lost. But repeat biopsies are invasive, sometimes sample only one disease site, and can miss the spatial and temporal heterogeneity of clonal evolution. That's the clinical gap a plasma-based assay is being asked to fill: can we monitor disease dynamics, and, ideally, the emergence of resistance, from a tube of blood rather than a needle in a node?

What's measured, and how

Here precision matters, because the phrase "CD20 ctDNA testing" invites a misunderstanding. Circulating tumor DNA (ctDNA) assays don't measure CD20 protein expression. They interrogate fragmented tumor-derived DNA shed into plasma, profiling somatic mutations, and, in some platforms, methylation signatures [1]. Loss of CD20 as an antigen is a protein-level phenomenon that IHC and flow cytometry are designed to catch; plasma sequencing can, at best, detect the genomic and clonal correlates of that process, such as mutations associated with clonal evolution after anti-CD20 exposure [1]. So a plasma assay isn't a drop-in replacement for a CD20 stain. It's a different lens on the same disease, better suited to tracking clonal dynamics over time than to directly certifying whether a given cell still displays the antigen.

The analyte, then, is cell-free tumor DNA; the assays are next-generation sequencing panels (often with error-correction chemistry), methylation profiling, and digital PCR. Sensitivity is the central technical challenge. ctDNA in lymphoma can be shed at low levels, and that fraction may fall further precisely when we most want to detect residual disease, after effective anti-CD20 treatment, when tumor burden is minimal. A 2024 review of sensitivity-improvement strategies catalogs the current toolkit for pushing the limit of detection lower: error-corrected sequencing to suppress background noise, methylation-based approaches that exploit the density of informative CpG sites, and digital PCR for tracking known variants [3]. These matter because a low-tumor-fraction plasma sample sits right at the edge of what today's assays can reliably call, and a false-negative in the MRD setting isn't a harmless miss.

The evidence

The framework for using ctDNA this way in lymphoma is now reasonably well articulated, even if the CD20-specific application remains early. A 2023 review positioned clinical ctDNA testing as a precision-oncology tool spanning both hematologic and solid malignancies, with explicit lymphoma applications, including the concept of using circulating tumor DNA mutation profiling to detect the clonal evolution associated with CD20 loss after anti-CD20 therapy, rather than reaching for a repeat tissue biopsy [1]. That's the conceptual scaffold: plasma as a serial, minimally invasive window on resistance biology.

More recently, the 2025 ASCO Educational Book reviewed liquid-biopsy approaches for cancer characterization, residual-disease detection, and therapy monitoring, again including lymphoma ctDNA utility [2]. The evidence summarized there supports plasma ctDNA for detecting residual disease and for monitoring disease dynamics, which is directly relevant to the period after anti-CD20 therapy, when CD20 expression may be shifting [2]. An honest word on what these sources are and aren't. They're reviews and educational syntheses establishing plausibility and framing, not key trials demonstrating that a plasma CD20-loss signal changes outcomes. The evidence suggests ctDNA can track lymphoma disease dynamics; it does not yet establish a validated plasma assay that certifies CD20 status or directs a change in the anti-CD20 drug class. That distinction is the whole ballgame for clinical adoption.

The state of the evidence

Regulatory reality anchors the honest answer: this is investigational. A 2023 perspective on the regulatory implications of ctDNA in immuno-oncology laid out frameworks emerging for solid tumors and noted that lymphoma, and CD20 liquid biopsy specifically, lacks FDA-approved companion-diagnostic status [4]; the vault likewise records no CD20 ctDNA companion diagnostic, and B-cell ctDNA-MRD sits at T1-pending. There is, at present, no plasma assay cleared to serve as a companion diagnostic for anti-CD20 therapies, and the pathway to that endorsement, analytical validation, clinical validation against outcomes, prospective evidence, remains to be walked [4]. Tissue-based IHC and flow cytometry stay the standard for determining CD20 expression.

So where does that leave the practicing oncologist or pathologist? ctDNA in lymphoma is best understood today as an investigational monitoring tool with genuine promise for MRD-guided thinking and for surfacing clonal evolution non-invasively [1][2]. The sensitivity work is active and necessary, because the low-tumor-fraction post-treatment sample is exactly where the assays are stressed [3]. And the regulatory framework for turning any of this into a companion diagnostic for the anti-CD20 class hasn't been established [4].

The measured take: plasma ctDNA may become a valuable complement to tissue for tracking lymphoma after anti-CD20 therapy, flagging when a disease is evolving and prompting the tissue confirmation that still governs CD20-directed decisions. It is not yet a validated substitute for that tissue, it does not directly read CD20 protein, and it should not, on its own, drive changes to a patient's therapy. This is a fast-moving space; the framing here reflects a snapshot, and the evidence base is still being built.

---

References

1. Park KU et al. Clinical Circulating Tumor DNA Testing for Precision Oncology. Cancer Research and Treatment. 2023. PMC10101787.

2. ASCO Education Committee. Liquid Biopsy Approaches for Cancer Characterization, Residual Disease Detection, and Therapy Monitoring. ASCO Educational Book (ascopubs.org). 2025. DOI: 10.1200/EDBK-25-481114.

3. [Resolve and insert the verified author byline from the published article before publication.] Improvement of the sensitivity of circulating tumor DNA-based liquid biopsy: current approaches and future perspectives. Exploration of Targeted Anti-tumor Therapy. 2024. explorationpub-1002333.

4. Authors per JITC publication. Regulatory implications of ctDNA in immuno-oncology for solid tumors. Journal for ImmunoTherapy of Cancer, 11(2):e005344. 2023. jitc-e005344.

5. National Complete Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines): B-Cell Lymphomas, immunophenotype/antigen notes (CD20 as anti-CD20 therapeutic target; CD20-expression states including loss). NCCN.