cfDNA-Based MRD Assay Is Feasible for Use in Relapsed/Refractory CLL

An analysis from the phase 2 CLL2-BAAG trial (NCT03787264) showed that cell-free DNA (cfDNA)–based minimal residual disease (MRD) testing was feasible in patients with chronic lymphocytic leukemia (CLL) who were treated with time-limited venetoclax (Venclexta), acalabrutinib (Calquence), and obinutuzumab (Gazyva).¹

Results presented during the 2021 International Workshop on Chronic Lymphocytic Leukemia showed that the cfDNA assay also had results that were highly concordant with 4-color flow cytometry (FC). Additionally, the cfDNA-based method was identified as a less expensive and quicker option that can complement FC when the 2 are used in combination, to improve MRD detection in this patient population.

“cfDNA seems to more accurately reflect the CLL burden in the whole body as opposed to the blood only, and the methods seem to have the biggest advantages compared with [FC] in patients with residual lymph nodes and less circulating cells, which is kind of what we expected but hasn’t been shown before,” said lead study author Moritz Fürstenau, MD, of the Division of Hematology, Immunology, Infectiology, Intensive Care, and Oncology at the University Hospital of Cologne in Germany, in a virtual presentation during the meeting.

Undetectable MRD (uMRD) status is a predictive factor of improved survival outcomes for patients with CLL. However, conventional MRD assessments through FC have demonstrated shortcomings, as seen in a pooled analysis.² Furthermore, patients with CLL who have residual disease in the lymph node compartment have poorer outcomes, despite reaching uMRD status in the peripheral blood and bone marrow, highlighting the unmet need that FC to assess MRD does not reflect residual disease across all compartments.

In the multicenter, investigator-initiated CLL2-BAAG trial, conducted by the German CLL Study Group, researchers enrolled 46 patients with relapsed/refractory CLL who received debulking treatment with bendamustine followed by 1 cycle of obinutuzumab alone before acalabrutinib was added at cycle 2 and a ramp-up schedule of venetoclax was started at cycle 3. Treatment continued until the maintenance phase and was guided by MRD; overall, it lasted between 14 and 34 months.

MRD assessments were utilized by both 4-color FC and circulating tumor (ct) DNA droplet digital polymerase chain reaction (ddPCR). Results showed that as treatment continued from cycle 5 into the maintenance phase, higher rates of uMRD were detected and varied between the 2 assays.

On day 1 of cycle 5, MRD-positivity rates were 60% and 53% in ctDNA ddPCR and FC, respectively, before decreasing to 44% and 36% at day 1 of cycle 6, respectively. At the interim stage, the MRD-positivity rate was 38% with ctDNA ddPCR and 31% with FC.

At the final restaging time point, Fürstenau noted that uMRD rates were highest at 80% with ctDNA ddPCR and 76% with FC; MRD-positivity rates were 16% and 22%, respectively. In the first maintenance cycle, the MRD-positivity and uMRD rates were 16% and 78%, respectively, with both assays. However, in the second maintenance cycle, MRD-positivity rates were 25% and 15% with ctDNA ddPCR and FC, respectively, and uMRD rates were 65% and 75%, respectively.MRD levels were more closely evaluated between the 2 assays.

“In cycle 1, so after 3 doses of obinutuzumab,the decrease in MRD is much steeper in the [FC] curve, which we explain is the effect of anti-CD20 antibody alone,” said Fürstenau. “In cycle 2, acalabrutinib is started and quite impressively we see a rapid increase in the median MRD and the [FC] curve almost back to baseline levels.” Fürstenau noted that although FC mirrors circulating disease only, cfDNA appears to reflect the entire disease burden.

Investigators also sought to question whether MRD detection was improved by combining the 2 methods in this trial. When examining 384 matching cfDNA and FC sam-ples, 308 of them (80.2%) showed concordant results. It was most prominent that cfDNA and FC both detected MRD (n = 199) and both detected no MRD (n = 109). Forty-four of 153 samples (28.8%) were detected as having MRD positivity via cfDNA but were previouslyclassified as uMRD by FC; cfDNA did not detect MRD in 31 samples that FC did.

“We can state that the 2 methods generated have very concordant results,” said Fürstenau. “The cfDNA-based method has produced highly concordant results compared with the gold standard of [FC], and if we try to quantify the benefit of adding cfDNA to our conventional [FC] setup, we see that 44 samples that were assessed as MRD negative by [FC] actually still had detectable CLL DNA fragments.”

Discordant results were also investigated in the study. Advantages for cfDNA were reported for patients with low absolute lymphocyte count (P = .009), residual lymphadenopathy (P = .003), and lactate dehydrogenase levels above 200 U/L (P = .027), suggesting that “cfDNA performs particularly well compared with conventional flow in patients with less circulating CLL cells and rather nodal disease.” Fürstenau concluded that patients on the trial will continue to be sampled for MRD in the maintenance phase, and future research will aim to determine how accurately cfDNA-based methods will predict duration of remissions to treatment and detections of nodal vs leukemic relapses.

_REFERENCES

_{1. Fürstenau M, Weiss J, Giza A, et al. Cell-free DNA (cfDNA)-based serial minimal residual disease assessment in patients with chronic lymphocytic leukemia treated with time-limited obinutuzumab, acal-abrutinib and venetoclax. Presented at: 2021 International Workshop on CLL; September 17-20, 2021; virtual. Abstract 1084129.}

_{2. Fürstenau M, Bahlo J, Fink AM, et al. Residual abdominal lymphade-nopathy after intensive frontline chemoimmunotherapy is associated with inferior outcome independently of minimal residual disease sta-tus in chronic lymphocytic leukemia. Leukemia. 2020;34(3):924-928. doi:10.1038/s41375-019-0597-3}