Novel Cell-Free DNA Assays Expand the Reach of MRD Detection in Lymphoma

Advancements in minimal residual disease (MRD) detection over the past decade have not only resulted in more sensitive cell-free DNA assays but have extended their utility across lymphoma subtypes, according to Reid W. Merryman, MD.¹

“The concept of MRD in lymphoma is not new. [MRD assays, such as] flow cytometry and polymerase chain reaction, have been used for decades, but only in lymphoma subtypes with a circulating component or chromosomal rearrangement. Advances in high throughput sequencing have allowed for the broader use of MRD using circulating tumor DNA [ctDNA] across all lymphoma subtypes,” said Merryman in his presentation during the 12th Annual Meeting of the Society of Hematologic Oncology. Merryman is an instructor in medicine at Harvard Medical School and an attending physician at Dana-Farber Cancer Institute in Boston, Massachusetts.

Merryman added that the common theme of this development has been that “more tumor reporters lead to improved sensitivity.”

Using diffuse large B-cell lymphoma (DLBCL) as a lymphoma focal point, Merryman discussed 3 categories of MRD assays: immunoglobulin high throughput sequencing (IgHTS), such as the clonoSEQ assay; panel-based assays, such as cancer personalized profiling by deep sequencing (CAPP-seq); and emerging assays, such as phased variant enrichment and detection sequencing (PhasED-seq) and minor-allele-enriched sequencing through recognition oligonucleotides (MAESTRO).

IgHTS

IgHTS are the least sensitive of the assay types, tracking 1 to 5 tumor reporters on average. These assays identifies and “tracks tumor-specific clonotypes,” explained Merryman.

“There have been a number of studies in DLBCL that have shown that ctDNA assessments using this approach are associated with disease bulk and dynamic changes over time have been associated with PET response and progression-free survival,” said Merryman. He added that a limitation of IgHTS is a modest lead time of just under 4 months. Lead time is the time from initial ctDNA detection to clinical relapse.

“If you had an assay that you’re hoping to use to guide therapy, you want…ideally a longer lead time to facilitate clinical implementation,” said Merryman.

Panel-based assays

The next category Merryman addressed was panel-based assays, which track dozens of tumor reporters. “These are disease-specific panels of mutations that facilitate tracking dozens of mutations with improved sensitivity compared to [IgHTS],” said Merryman.

Merryman highlighted a study that compared CAPP-Seq, a type of panel-based assay, with immunoglobulin sequencing in patients with DLBCL.² According to Merryman, the study showed that CAPP-Seq was associated with “a higher likelihood of being able to identify a tumor genotype, higher likelihood of identifying pretreatment ctDNA, and also a higher likelihood of identifying ctDNA at the time of relapse compared to IgHTS.”

A separate study of CAPP-Seq in DLBCL demonstrated that pretreatment ctDNA levels were prognostic of patient outcomes.3 Building on these findings, researchers further explored panel-based assays using plasma samples from the phase 3 POLARIX trial [NCT03274492] of treatment naïve patients with DLBCL.⁴ This analysis fell short, according to Merryman, because the prognostic value of the assays was shown to be best at the end of the trial. The optimal time for the best prognostic value is early on, when treatment can still be affected. Another issue that emerged was that 3-year rates for PFS were only modestly better in patients with negative ctDNA at 81% vs 51% in patients with positive ctDNA.¹

Newest assays

Researchers are now looking to build on these panel-based assay results with newer assays that “maximize tumor reporters by using phased variants or whole genome sequencing (WGS) and are often able to track hundreds or even thousands of tumor reporters to maximize sensitivity,” said Merryman.

“PhasED-seq is a newer ctDNA assay that takes advantage of phase variants. Phase variants occur when multiple mutations occur on a single DNA molecule in close proximity, usually within about 170 base pairs,” according to Merryman. “So traditionally, we rely upon duplex sequencing, which requires independent recovery of both strands of DNA to guard against false positives, but recovery of a single strand of DNA is sufficient for identification of phase variants without a risk of false positivity.” Research has shown that PhasED-seq has improved sensitivity compared with CAPP-Seq.⁵

Another novel MRD assay being explored in lymphoma is the whole-genome sequencing MAESTRO assay, which “uses short allele-specific probes to enrich thousands of prespecified mutations and enable their detection using Duplex Sequencing with up to 100-fold fewer sequencing read,” according to Merryman. A pilot study has shown high sensitivity of MAESTRO as a marker of relapse in patients with DLBCL.⁶

In his concluding remarks, Merryman said, “Novel assays have improved the sensitivity of ctDNA detection in lymphoma, and with these improvements, we're now seeing multiple clinical trials that are actually adapting therapy based on the ctDNA results.”

References

1. Merryman RW. Role of MRD in lymphoma — who, what, when, and how? Presented at: 12th Annual Meeting of the Society of Hematologic Oncology. September 4-7, 2024. Houston, TX.
2. Scherer F, Kurtz DM, Newman AM et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA.Sci Transl Med. 2016;8(364):364ra155.doi:10.1126/scitranslmed.aai8545
3. Kurtz DM, Scherer F, Jin MC, et al. Circulating Tumor DNA measurements as early outcome predictors in diffuse large B-cell lymphoma. J Clin Oncol. 2018;36(28):2845-2853. doi:10.1200/JCO.2018.78.5246
4. Herrera AF, Tracy S, Sehn LH, et al. Circulating tumor DNA (ctDNA) status and clinical outcomes in patients (pts) with previously untreated diffuse large B-cell lymphoma (DLBCL) in the POLARIX study. J Clin Oncol;41(suppl 16):7523. doi:10.1200/JCO.2023.41.16_suppl.7523
5. Kurtz DM, Soo J, Keh LCT, et al. Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA. Nat Biotechnol. 2021;39(12):1537-1547. doi:10.1038/s41587-021-00981-w
6. Merryman RW, Rhoades J, Xiong K, et al. Comparison of whole-genome and immunoglobulin-based circulating tumor DNA assays in diffuse large B-cell lymphoma.Hemasphere. 2024;8(4):e47.doi:10.1002/hem3.47