At Increasing Levels of Sensitivity, NGS-MRD Detects CLL in Bone Marrow

AJMC Staff

A study presented at the 61st American Society of Hematology (ASH) Annual Meeting and Exposition in Orlando, Florida, discussed the first report of next-generation sequencing (NGS) to test for minimal disease response (MRD) after the combination of ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) in chronic lymphocytic leukemia (CLL).

Chemoimmunotherapy (CIT) era and venetoclax have shown the correlation between MRD measured by at least 4-color flow cytometry (FC), and progression-free (PFS) and overall survival (OS) in CLL. But the ability to achieve sustained undetectable MRD (uMRD) remission is lacking for the majority of patients with CLL treated with these regimens.

The expanded MRD analysis used standard flow cytometry and assessed using NGS-MRD, focusing on mid-FCR (C3) and 2 month post-FCR (EOT) timepoints. The  NGS-MRD assay targets immunoglobulin receptor sequencing with up to 10E-6 sensitivity for detection of B-cell malignancies.

iFCR is a multicenter single-arm phase 2 trial at 7 sites across the United States; 85 patients aged 65 years or younger with previously untreated CLL were enrolled and treated with iFCR.

Per protocol analyses of MRD in both peripheral blood (PB) and bone marrow by standard 4-color FC were performed at local laboratories at C3. Both PB and BM samples were sent for NGS-MRD evaluation at end of therapy (EOT).

Forty-eight patients had paired bone marrow and PB samples with 16 additional PB only samples. NGS-MRD status was evaluated at 10E-5 and 10E-6 levels, and defined as positive if 1 or more rearrangement was detected per 100,000 or per million cells, respectively. An indeterminate finding was reported if insufficient cells were assayed.

At the C3 restage, the bone marrow-uMRD rate by flow was 47%, with 100% concordance to flow PB-uMRD status in all patients with bone marrow-uMRD. However, 33% (12/36 evaluable) with detectable cells in marrow had PB-uMRD, demonstrating enhanced sensitivity of bone marrow-MRD testing.

At EOT, bone marrow-uMRD rates rose to 78%, compared with 86% in PB, including 14/24 patients converted from bone marrow-positive/PB-positive to bone marrow-negative/PB-negative and 7/12 bone marrow-positive/PB-negative to bone marrow-negative/PB-negative.

In NGS-MRD analysis from 48 patients with evaluable bone marrow and PB samples at EOT, a larger number of patients were MRD positive in bone marrow (n = 21; 43.8%) vs. PB ( n = 13; 27.1%) (McNemar test: P = .04).

Detection of residual disease in both bone marrow and PB improved with increasing sensitivity, with greater detection in bone marrow. There was 54% positive at 10E-6 sensitivity in this cohort, compared with 36% in PB.

Evaluation for true negative samples at 10E-6 sensitivity was indeterminate, hence definite uMRD was seen in only 23% bone marrow and 9% PB.

Fifty-two patients with PB-uMRD by FC at EOT had associated PB NGS-MRD results: 10 PB-uMRD by FC were positive at 10E-5 with 8 additional positive at 10E-6 (35% greater than FC).

In bone marrow, similar results were observed: of 44 patients with bone marrow-uMRD by FC at EOT, 13 were positive at 10E-5 with 9 additional positive at 10E-6 by NGS-MRD (50% greater than FC).

When this higher sensitivity bone marrow-uMRD data is used to define overall clinical response at EOT, the CR/CRi with bone marrow-uMRD rate at 10E-5 is 32.6% (14/43), and at 10E-6 is 16.2% (6/37), compared to 43.8% (21/48) using 4-color FC.

The rate of BM-uMRD would be 60.5% (26/43) at 10E-5 sensitivity and 29.7% (11/37) at 10E-6, with NGS-MRD.

In summary, testing after iFCR demonstrates that 50% of patients with bone marrow-uMRD by flow cytometry have detectable CLL cells at the level of detection of 1 or greater per million cells; these findings suggest that CLL cells are still frequently present.

Longer follow-up is needed to correlate MRD with PFS in this setting, and future studies should incorporate NGS-MRD assessment with larger volume cell sampling to ensure adequate sensitivity and evaluate venetoclax-based regimens, the authors said.


Vartanov AR, Fernandes SM, Nguy WI. et al .High sensitivity NGS analysis of MRD in CLL patients prospectively treated with Ibrutinib plus FCR (iFCR). Presented at the 61st American Society of Hematology Annual Meeting and Exposition, Orlando, Florida; December 7-10, 2019; Poster 4291.
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