P-068: IMPLEMENTATION OF NEXT GENERATION SEQUENCING AS BEST TOOL FOR ROUTINE EVALUATION OF MINIMAL RESIDUAL DISEASE IN THE DAILY PRACTICE OF MULTIPLE MYELOMA PATIENTS

Introduction: In recent years, minimal residual disease (MRD) reduction up to undetectable level has become an independent prognostic factor in Multiple Myeloma (MM) and MRD assessment might become a helpful tool to guide treatment.
Here we present the implementation of a highly sensitive Next Generation Sequencing (NGS) workflow for MRD assessment in MM daily clinical practice.

Methods: 299 newly diagnosed transplant-eligible MM patients (pts) were included in the study between 2016 and 2023, and screened by NGS for clonotype (ID) definition. MRD was assessed pre-maintenance, at the achievement of at least very good partial response, and, thereafter, once a year. A commercial assay covering IgH and IgK genes (Invivoscribe) was employed for ID definition in bone marrow (BM) CD138+ plasma cells (PCs), and for MRD measurement in BM whole blood cells. Data were analyzed by Lymphotrack Dx and MRD software (Invivoscribe).

Results: ID screening was possible in all pts, by using 70ng of starting DNA and by sequencing firstly IgH-FR1 and Igk and, in cases of no ID identification, also IgH-FR2 and -FR3. Overall, 490 clonal sequences were identified in 281/299 pts (94%). Polyclonal results (6%) were mainly due to low CD138+ PCs percentage in pre (˂1.6%) and post ( < 84%) enrichments, as well as to low cells number (p-value ˂.05).
MRD trackable sequences were selected thanks to an originality score defined according to V and J regions specificities, CDR3 insertions and/or deletions and clonality percentage. Overall, 246 IgH (177 FR1, 43 FR2 and 26 FR3) and 35 IgK IDs were selected, with a median clonality percentage of 64.5% (2.5%-98.6%). In 10 pts, 2 IDs were identified and used for MRD monitoring, to improve MRD analysis’ sensitivity.
For MRD detection, 2µg input DNA’s triplicates were sequenced with a 1,5 million reads depth. A confidence ≥90% was required to define the sensitivity level. So far, 317 MRD measurements were evaluated in 111/281 pts, (at least one per pts), with an overall median follow-up of 47 months (range 6-83). MRD results were defined negative, with 10-5 sensitivity in 80% of cases, in 146/317 cases; positive in 117/317 cases, with MRD ranging from 1.1x10-5 to 2.3x10-4; low positive (when measurements had < 90% result’s confidence) in 54/317 cases, with MRD ranging from 9.9x10-9 to 9,8x10-5. In 50/111 pts, MRD was also measured pre-maintenance (67 evaluations), with 24/67 MRD negative results.

Conclusions: In conclusion, NGS was successfully implemented as best tool in the daily practice of MM pts. The clonotype was defined in almost all pts, thus allowing MRD molecular monitoring. CD138+ PCs’ both quality and amount were critical for successful clonotyping. For MRD assessment, a regular samples’ workflow (at least 5 per week) was needed to both limit costs and maintain an acceptable reporting time (7 days). Results confirm the feasibility of MRD assessment by NGS in the daily practice, supporting the upcoming need of MM pts personalized management.
Acknowledgment: AIRC IG2018-22059