Representativeness of clone size measurement by using NGS-based immunoglobulin gene rearrangement test in comparison to flow cytometry in clonal plasma cell disorders Free

Introduction: Next-generation sequencing (NGS) IG gene rearrangement test provides quantitative information about the clone, which helps monitor residual disease in clonal plasma cell disorders; however, discrepancies between flow cytometry (FCM) and NGS have been reported at the minimal residual disease (MRD) level. We compared the clone size at initial diagnosis to confirm the intrinsic difference resulting from each method for the first time using all 4 IGH primer sets and IGK primer set. We also compared the IG gene usage information, which is associated with the clinico-pathologic characteristics, with previous Western studies.

Methods: The cohort included 58 patients diagnosed with clonal plasma cell disorders at Asan Medical Center in Seoul, Republic of Korea, from March 2023 to September 2024, with plasma cell myeloma being the most common diagnosis (79.3%). For flow cytometry (FCM), clonal plasma cells were identified by the absence of CD19 and CD45 expressions and the presence of CD56. For NGS, primary detection used IGH FR1 and IGK primer sets simultaneously. If IGH FR1 fails to detect a clonal IGH rearrangement, additional primer sets—IGH leader, IGH FR2, and IGH FR3—were utilized. Clone size quantification for NGS defined “IGH_max” as the largest clone size from all IGH primer sets and “IG_max” as the largest clone size considering both IGH and IGK primer sets. The clone size was expressed as the proportion of total B-cells. IG gene usage profiles were compared to three Western studies.

Results: Among of the 58 patients who underwent NGS, 49 had FCM results, and all showed clonal plasma cell populations. The clonal cell percentage detected by FCM was 67.23% (range 0.88–100.00%). NGS identified clonal IGH rearrangements in 42 patients (72.4%), of which 23 were detected using the IGH FR1 primer set (23/49, 46.9%). We tested IGH leader, FR2, and FR3 primer sets in 21, 22, and 22 patients, respectively among those 26 who did not show clonal IGH rearrangements using the IGH FR1 primer set: IGH leader, FR2, and FR3 showed clonal IGH rearrangements in 8 (38.1%), 7 (31.8%), and 6 (27.3%) patients, respectively, which increased the detection of clonal IGH rearrangements in 31 patients (31/49, 63.3%). The clonal cell percentage of IGH_max was 9.4% (range 0.00-95.6%, median [range]). The IGK primer showed a higher detection rate of 71.4% (35/49).ClonalIGK rearrangement was observed in 15 patients who did not show clonalIGH rearrangement using the IGH FR1 primer set. The clonal cell percentage detected with the IGK primer set was 19.9% (0.00-94.1%). Overall, NGS detected clonal IGH or IGK gene rearrangements in 38 patients (38/49, 77.6%), and the IG_max was 28.4% (0.0-95.6%). The Bland-Altman plot analysis showed that clone size measurements from FCM were larger than those from NGS: the mean difference was 39.9% in IGH_max, 29.2% in IGK, and 24.1% in IG_max.

The IG gene usage profiles and the comparison with the three previously published Western studies (Medina et al. 2020, Ferroro et al. 2012, Hadzidimitriou et al 2006.) revealed unique ethnic differences: IGHV gene subgroup-level comparison showed IGHV2-70, IGHV3-11, IGHV3-13, and IGHV3-15 were more common in Koreans (9.5% vs 2.2%; P=0.027, 9.5% vs 2.0%; P=0.023, 4.8% vs 0.3%; P=0.032, and 9.5% vs 2.5%; P=0.036, respectively);IGHJ5 was found to be more frequent in Koreans (21.4% vs. 10.0%; P = 0.033). Regarding IGK rearrangement, only one previous study (Hadzidimitriou et al.) investigated its patterns. IGKV4-1 was less common in Koreans (9.2% vs. 22.5%), while Intron-KDE was more common (23.7% vs. 0%) in Korean patients compared to Western studies. Chi-squared test revealed a significant difference in the overall distribution of IGK rearrangement clones between Korean and Western patients. Comparison ofIGK rearrangement combinations showed that IGKV-J + 2 IGKJ-C-intron-KDE was significantly more common in the Korean population compared with Western data (9.3% vs. 0.0%; P = 0.014).

Conclusion: The detection of clonal rearrangement was lower in NGS than in FCM, both qualitatively and quantitatively, which might result in the discrepancies between the two methods at the MRD level. Using IGK or other IGH primers alongside the IGH FR1 primer set showed additive benefits. Korean patients displayed a unique IG gene profile compared to Western groups, indicating ethnic differences inIG gene usage in plasma cell disorders.