Next generation sequencing (NGS) and ctDNA in lymphoma
Next generation sequencing(NGS) technology plays a pivotal role and understanding the molecular landscape in oncology by providing a comprehensive analysis of genomic mutations.
The detection and analysis of mutations have improved tumor evaluation, and NGS offers a multitude of diagnostic, prognostic and management options in caring for patients with cancer.
Circulating tumor DNA (ctDNA) is an emerging, minimally invasive biomarker with applications across multiple lymphoma subtypes, including baseline prognostication, treatment response assessment, minimal residual disease (MRD) detection, and early relapse prediction.
ctDNA profiling offers real-time genetic insights without the limitations of traditional, invasive tissue biopsies.
Circulating tumor DNA (ctDNA) is transforming the management of lymphomas by providing a minimally invasive “liquid biopsy” to track tumor burden, monitor response to therapies (such as CAR-T), and detect minimal residual disease (MRD).
ctDNA is increasingly incorporated into clinical trials and has been recognized as a potential adjunct to PET/CT.
Lymphomas are highly heterogeneous at the molecular level and this heterogeneity influences clinical behavior and treatment responses.
NGS technology can aid diagnosis, prognostication and targeted intervention by identifying high risk features , and complex karyotypes, and evolution throughout the disease course.
The incidence of malignant lymphoma has been steadily increasing, and is the most prevalent hematological malignancy among adults in the US.
Traditional prognostic methods rely on clinical and histopathologic features requiring the use of tissue biopsies, serologic tumor markers, imaging, immunohistochemical analysis, and fluorescence in situ hybridization studies.
In addition, ctDNA alongside other markers can aid in the measure residual disease (MRD to identify patient at risk for relapse.
Diffuse Large B-Cell Lymphoma (DLBCL)
DLBCL has the most robust evidence for ctDNA utility:
Pretreatment ctDNA levels correlate with tumor burden (IPI, LDH, total metabolic tumor volume) and are independently prognostic for event-free survival (EFS) and overall survival (OS).
Early molecular response: A 2-log reduction after 1 cycle (early molecular response, EMR) and a 2.5-log reduction after 2 cycles (major molecular response, MMR) are strongly associated with superior outcomes.
End-of-treatment MRD: In a pooled analysis of 137 patients from five prospective studies, end-of-therapy ctDNA-MRD had greater prognostic utility than PET.
Two-year PFS was 97% for MRD-negative vs. 29% for MRD-positive patients.
The DIRECT study confirmed these findings with 2-year time to progression of 95% vs. 42%.
Noninvasive genotyping: ctDNA enables molecular subtyping (cell of origin, genetic subtypes) without tissue biopsy, and captures clonal heterogeneity including mutations absent from tissue.
NCCN recognition: The NCCN B-Cell Lymphoma guidelines note that if end-of-treatment PET is positive, ctDNA-MRD assessment (category 2B) using a test with a detection limit of <1 part per million may be considered prior to additional therapy; if biopsy and/or ctDNA-MRD are negative, the PET-negative pathway may be followed.
Classical Hodgkin Lymphoma (cHL)
ctDNA is particularly valuable in cHL because Hodgkin/Reed-Sternberg cells constitute only ~5% of the tumor mass, making tissue-based genomic profiling challenging.
ctDNA mirrors HRS cell genetics, with STAT6, SOCS1, B2M, and TNFAIP3 among the most frequently mutated genes.
MRD after 2 cycles: In the GHSG HD21 trial, MRD-2 positivity by the LymphoVista assay predicted inferior 4-year PFS (36.7% vs. 82.2%; HR 5.3).
Combining MRD-2 with PET-2 identified patients at very low and very high risk of relapse.
Meta-analysis: A Bayesian meta-analysis of 1,158 patients showed progressively stronger prognostic associations from baseline (HR 2.74) to interim (HR 5.99) to end-of-treatment ctDNA positivity (HR 13.4) for PFS.
Follicular Lymphoma (FL)
Prediction of POD24: Approximately 20% of FL patients experience progression within 24 months (POD24), a high-risk event.
Higher baseline ctDNA levels are significantly associated with POD24 and non-CR.
In the GALLIUM trial, baseline ctDNA predicted shorter PFS and outperformed FLIPI for POD24 prediction (0.69 vs. 0.57).
Combined PET + ctDNA: In the RELEVANCE trial, combining PET response with ctDNA MRD at end of induction achieved a positive predictive value of 85.7% for POD24, compared to 58.3% for ctDNA alone and 45% for PET alone.
Longitudinal monitoring: ctDNA clearance at end of treatment correlates with durable remission, while persistent ctDNA identifies patients at high risk of early relapse.
ctDNA has shown particular promise in predicting outcomes after CAR T-cell therapy across multiple products:
Axicabtagene ciloleucel: In a prospective multicenter study, 70% of durable responders vs. only 13% of progressors had undetectable ctDNA 1 week after infusion.
Detectable ctDNA at day 28 predicted markedly inferior PFS and OS.
Lisocabtagene maraleucel: In the TRANSFORM trial, ctDNA clearance predicted longer EFS at all time points, with liso-cel achieving significantly more MRD-negative results than ASCT.
MRD status provided prognostic value beyond PET.
By weeks 2–4 post-infusion, responders approach 0 ppm ctDNA while nonresponders show persistence, enabling very early identification of treatment failure.
ctDNA re-emergence after initial clearance can predict relapse before radiographic detection, with ctDNA detected at or before relapse in 94% of progressing patients.
Mantle cell lymphoma (MCL): Undetectable MRD after induction may identify patients who can safely omit high-dose chemotherapy consolidation.
Primary CNS lymphoma (PCNSL): CSF ctDNA analysis shows superior sensitivity for CNS involvement compared to conventional imaging and cytology.
Marginal zone lymphoma: Early data show ctDNA detection rates of ~68% with potential for disease surveillance.
Primary Clinical Applications
Minimal Residual Disease (MRD) Detection: Serial ctDNA testing during or at the end of treatment is a powerful prognostic tool.
Rapid clearance of ctDNA indicates a strong likelihood of a sustained response, while detectable ctDNA highly correlates with a risk of relapse.
Non-Invasive Genotyping: Lymphoma cells rarely circulate freely in the blood, making plasma ctDNA testing a crucial tool for sequencing tumor-specific mutations (like $STAT6 or pathways like NF-\kappa B).
This helps doctors understand the tumor’s genetic profile without surgical excisions.
ctDNA assays provide quantitative insights that complement standard imaging, such as PET scans.
If PET scan is unclear, a negative ctDNA result can reduce the predicted risk of relapse, whereas a positive ctDNA test in a patient with a negative PET scan can act as a red flag for residual disease.
Lymphoma Subtypes
Diffuse Large B-Cell Lymphoma (DLBCL): Baseline and end-of-treatment ctDNA levels heavily correlate with survival outcomes. ctDNA tracking can expose genetic evolution, resistance to therapies, and early signs of relapse well before clinical symptoms appear.
Hodgkin Lymphoma (HL): Tumor cells make up only a tiny fraction of the mass in HL.
ctDNA assays can track tumor burden from the beginning of treatment and help identify patients who may be eligible for therapy de-escalation to reduce long-term toxicity.
Follicular Lymphoma (FL): Elevated baseline ctDNA is an independent prognostic factor and is significantly associated with shorter progression-free survival.