Comparison of MRD Detection in Autografts in Multiple Myeloma between Novel High-Sensitivity Euroflow-NGF and NGS
Authors: Urushihara Ryota , Takezako Naoki , Yoroidaka Takeshi , Yamashita Takeshi , Nakao Shinji , Takamatsu Hiroyuki 
Published: November 2021

Abstract
Background: Autologous stem cell transplantation (ASCT) remains the gold-standard treatment for multiple myeloma (MM). To date, we have reported the prognostic value of minimal residual disease (MRD) detection in autografts in an ASCT setting using EuroFlow next-generation flow (NGF) and next-generation sequencing (NGS) (Takamatsu et al., ASH 2018, 2020). The main problem with NGF is its lower sensitivity (2 × 10 -6) compared with that of NGS (&lt;1 × 10 -6).
Methods: We reanalyzed 11 autografts in which the MRD were negative on NGF but positive (n = 7) or negative (n = 4) on NGS (Takamatsu et al., ASH 2018, abstract #258) using 5-20 mL of autografts with NGF to increase the sensitivity of MRD detection. Additionally, we enrolled 9 patients with newly diagnosed MM, from whom 5-20 mL of apheresed autografts were cryopreserved. We included 20 patients with newly diagnosed MM. The median age at ASCT was 60 (range, 45-67) years, and the patients included 12 men and 8 women at International Staging System I (n = 3), II (n = 13), and III (n = 4), 6 of whom harbored high-risk chromosomal abnormalities, including t(4;14) (n = 3), t(14;16) (n = 1), del17p (n = 1), and t(4;14) and del 17p (n = 1). All patients received bortezomib-based chemotherapy for induction followed by melphalan at a dose of 200 mg/m 2 for conditioning before ASCT. Three patients received consolidation therapy with carfilzomib-lenalidomide-dexamethasone (n = 2) or bortezomib-lenalidomide-dexamethasone (n = 1), and 18 patients received lenalidomide (n = 16), thalidomide (n = 1), or thalidomide and lenalidomide (n = 1) maintenance. Frozen autografts (n = 20) were thawed for MRD assessment using NGF and NGS. The NGF method was based on a previous report (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD assay (Seattle, WA) (Ching et al., BMC Cancer 2020). The NGF method was modified to increase the sensitivity of MRD detection by capturing up to 6 × 10 7 cells.
Results: Frozen autografts were used in this study; therefore, we performed a sensitivity test using a dilution of frozen/thawed primary MM cells in an autograft with NGF. The sensitivity test revealed a strong correlation between 5 × 10 -7 and 1 × 10 -4 MRD levels (Figure 1A; r = 0.999, P &lt;0.0001). Next, MRD in autografts (n = 20) was evaluated using NGF and NGS. The sensitivity of NGS was 1.7 × 10 -7-8.7 × 10 -5 (median, 7 × 10 -7) using 2-4 mL of autografts; the sensitivity of NGF was 1.6 × 10 -7-3.7 × 10 -6 (median, 3 × 10 -7) using up to 20 mL of autografts based on the detection of ≥10 abnormal cells. MRD levels in autografts using NGF and NGS were correlated (Figure 1B; r = 0.979, P &lt;0.0001). There was no discrepancy in the MRD negativity between both methods except for two cases (MRD-negative on NGF/positive on NGS [n = 2]). All high-risk chromosomal abnormality cases (n = 6) revealed MRD levels &lt;10 -5 butonly three patients achieved MRD levels &lt;10 -6. The best responses included 16 cases of a stringent complete response and 4 of a very good partial response. Three patients with positive MRD in the autograft exhibited progression. With a median follow-up period of 41 months after ASCT, the 3-year progression-free survival (PFS) was 90%, and the 3-year overall survival was 95%. There was no significant difference in the PFS based on the MRD levels between NGF and NGS. MRD negative cases tended to show better PFS than MRD positive ones (Figure 1C; P = 0.077 by NGF; P = 0.111 by NGS).
Conclusion: The modified EuroFlow-NGF method may be used to assess MRD in frozen/thawed autografts, and its sensitivity may increase up to 5 × 10 -7, which is comparable to that of NGS.
Figure 1 Figure 1.

Disclosures
Yamashita: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; celgene: Honoraria; Takeda: Honoraria. Nakao: Novartis Pharma: Honoraria; Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion Pharma: Research Funding. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.