Predictive Value of Next-Generation Sequencing-Based Minimal Residual Disease after CAR-T Cell Therapy
Authors: Huang Yue , Zhao Houli , Shao Mi , Zhou Linghui , Wei Guoqing , Wu Wenjun , Chang Alex Hong , Sun Tao , HU Yongxian , Huang He 
Published: November 2021

Abstract
Background
Minimal residual disease (MRD) is closely associated with risk stratification of hematological malignancies. Monitoring the MRD levels of patients during treatment and at time points after remission is critical for prevention of relapse. Chimeric antigen receptor T (CAR-T) cell therapy redirects genetically modified immune cells to fight against hematologic malignancies. However, given the high relapse rate after CAR-T cell therapy, MRD monitoring by traditional techniques cannot accurately quantify the disease burden, nor can they perform high-sensitivity in-depth monitoring. Further treatment options are still controversial at such a time point when the flow cytometry (FC) results show MRD &lt; 0.01%, while tumor clones remain. Next-generation sequencing (NGS) - MRD screens out patient-specific T/B cell receptors and accurately and quantitatively monitor patient-specific tumor cells (up to 10E-6) to reveal accurate and highly sensitive MRD levels, plus provide more timely intervention criteria.
Method
Between June 2016 and June 2020, we retrospectively enrolled 27 patients who achieved complete remission at the first 4-week evaluation after CAR-T cell therapy. BM samples were harvested and stored before CAR-T cell infusion and 10-154 days after infusion. We evaluated 63 specimens in total 27 patients, plus tracked immunoglobulin (IG) sequencing rearrangement by next-generation sequencing (NGS) in 20 patients. Next, we classified 17 patients into high-risk (HR, NGS-MRD positive) and low-risk (LR, NGS-MRD negative) groups according to the lower limit of NGS detection (10E-6), as well as performed leukemia-free survival (LFS) and overall survival (OS) analysis.
Results
At least one trackable IG clonal sequence was identified in the pre-CAR-T BM specimens from 20/27 of the cases analyzed. The two diagnostic samples with low DNA quantity, in addition to five samples lacking a dominant index sequence were excluded from further analysis. We measured the MRD in 63 samples, using a threshold of 0.01%, the determination of the presence (or absence) of leukemia was concordant in 46/63 (73%) of the samples. Discordance between NGS and FC was identified in 17/63 samples (27%).
Of the 20 patients for whom trackable sequences were identified, 17 with detectable clonal index sequences on +30 days were included in our subsequent analysis cohort (Figure 1). The baseline characteristics of the patients are presented in Table 1. NGS identified 9 out of the 17 patients (52.9%) whose level of MRD was &gt; 0.01%, but MRD negative as measured by FC. This controversial group (n=9) had inferior LFS than those whose MRD was less than 0.01% by NGS (median, 56 days vs. 219 days, p = 0.037) (Figure 2A). The OS rate was comparable between the two groups (p = 0.129) (Figure 2B).
Patients with positive NGS-MRD at day 30 had comparable LFS compared with those with negative NGS-MRD (p = 0.103) (Figure 3A). For subgroup analysis, we further analyzed the influence of HSCT on prognosis of HR and LR patients. Of the total 11 patients in HR group, seven non-HSCT subjects all relapsed within three months, with a median LFS of 38 days. In contrast, the remaining four HSCT patients in HR subgroup had a significantly better LFS than the non-HSCT patients (median, 495 days vs. 38 days, p = 0.003) (Figure 3B). These four patients also exhibited better OS to that of the non-HSCT group (median, 768 days vs 409 days, p = 0.006) (Figure 3C). As for the LR cohort, no significant difference was found in LFS and OS between the HSCT and non-HSCT groups (LFS: p = 0.782, OS: p = 0.782) (Median LFS and OS data not shown). All of the evidence demonstrated that NGS-MRD early after CAR-T is an efficient prognostic factor, especially for early relapse prediction. Early post-CAR-T NGS-MRD status may be recommended for HSCT timing. As such, for HR patients, whose NGS-MRD results were positive, timely HSCT may significantly improves the prognosis and therefore is highly recommended at an early point after CAR-T cell therapy.
Conclusion
NGS-MRD elaborately demonstrated the tumor dynamics. NGS-MRD evaluated early after CAR-T cell therapy is an efficient prognostic factor, and timely HSCT is highly recommended for NGS-MRD positive patients at an early point after CAR-T cell therapy.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.