Integrating next-generation sequencing disease evaluation and PCR based CAR-T cell tracking to guide decision making in a real-world cohort of pediatric B-cell ALL patients. Free

Background: Chimeric antigen receptor T-cell (CAR-T) therapy is an effective treatment option for relapsed/refractory pediatric B-cell acute lymphoblastic leukemia (B-ALL). A thorough understanding of both disease status and CAR-T cell kinetics is required to optimize patient outcomes. Here we report our multi-center experience with the use of next-generation sequencing (NGS) disease assessment and PCR-based CAR-T cell tracking to inform post-infusion management.

Methods: Pediatric B-ALL patients who underwent CAR-T therapy with tisagenlecleucel (tisa-cel) were retrospectively analyzed. Minimal residual disease (MRD) was serially assessed via multiparametric flow cytometry (MFC) and NGS evaluation of immunoglobulin heavy chain (IgH) rearrangements (clonoSEQ®, Adaptive Biotechnologies). CAR-T cell persistence was monitored via standard assessment of B cell aplasia (BCA, <50 CD19⁺ cells/μl)and quantitative PCR tracking (ExPeCT™ assay, Eurofins Viracor) in peripheral blood. Overall survival (OS) was defined as the time from CAR-T infusion to death of any cause. Event-free survival (EFS) was defined as survival without emergence of MFC⁺ MRD and censored at allogeneic hematopoietic cell transplant (allo-HCT).

Results: Forty-eight consecutive patients with a median age of 13 years (range 0-25) were analyzed. Fourteen patients (29.2%) had primary refractory disease, 23 patients (47.9%) were in first and 11 patients (22.9%) in second or greater relapse. Median baseline bone marrow (BM) disease burden by MFC was 0.1% (range 0%-97%).

Of 46 evaluable patients, 33 (71.7%) had an MFC⁻NGS⁻complete remission (CR) at day 30 post-infusion. The following outcomes were observed in these patients: 16 (48.5%) patients remain in remission, 8 (24.2%) underwent consolidative allo-HCT pre-emptively (n=3) or for early B cell recovery (BCR, ≥50 CD19⁺ cells/μl) with (n=1) and without (n=4) emergence of NGS⁺ MRD, and 9 patients (27.3%) suffered disease relapse at a median of 217 days (range 62-280) from CAR infusion (CD19⁻relapse, n=5; CD19⁺relapse, n=4). Of 13 patients not in NGS⁻CR at day 30, four underwent consolidative allo-HCT, 3 underwent repeat tisa-cel infusion, 2 received investigational CD22 CAR-T therapy, one became NGS⁻ without further intervention and 3 patients experienced CD19⁻relapse at a median of 88 days post-infusion (range 59-99). With a median follow up of 288 days (range 8-988), 6-month OS and EFS were 90.7% (95% CI 76.9%-96.4%) and 73.0% (95% CI 56.4%-84.1%) for the entire cohort, respectively.

Median CAR transgene levels at days 7, 14, and 30 were 28,560 (range 0-971,800), 8,399 (range 75.1-396,600) and 1,296 (range 5.47-141,400) vector copies/μg DNA, respectively. Median day 14 vector copies/μg DNA were significantly higher in patients with >5% BM blasts at baseline (50,820 vs. 5,664, p=0.0138), a CAR-T cell dose of >3x10⁶/kg (16,800 vs. 4,128, p=0.0061) and grade ≥2 CRS (100,655 vs. 5,297, p=0.0003). Among 17 patients who experienced BCR, median transgene levels were 27.07 (range 0-255.9) vector copies/μg DNA at that time. In contrast, a separate cohort of 6 long-term survivors of ≥ 4 years with ongoing BCA demonstrated a median of 220.7 (range 48.1-387.1) vector copies/μg DNA at last follow-up.

To capture early markers of potential treatment failure, we analyzed stringent event-free survival (S-EFS) in NGSMRD negative responders with available day 30 transgene data (n=28). S-EFS was defined as time from CAR-T infusion to either early BCR (<180 days), re-emergence of any NGS/MFC MRD or death and censored at pre-emptive allo-HCT (n=3) or scheduled tisa-cel reinfusion as part of an investigational protocol (n=2). In 14 patients with high CAR transgene levels (≥ 1200 vector copies/μg DNA) at day 30 post-infusion, 6-month S-EFS was 57.4% (95% CI 20.5%-82.4%), compared to 16.9% (95% CI 2.7%-41.7%) in 14 patients with lower transgene levels (p=0.0378).

Conclusions: NGS testing plays an important role in the response monitoring of pediatric B-ALL patients undergoing CAR-T therapy. Patients who remain NGS positive beyond day 28 typically require further intervention. Even among patients achieving NGSnegative remission, approximately 40% may require additional treatment due to relapse and/or early BCR. Quantitative PCR tracking of CAR-T cells could potentially serve as a valuable tool in identifying these patients at risk for treatment failure and validation of these findings in a larger patient cohort is warranted.