A novel in-house deep sequencing method for non-invasive disease monitoring in multiple myeloma patients

Background: Novel and more effective treatment strategies have sig- nificantly prolonged multiple myeloma (MM) survival and raised inter- est in the depth of response. This implies the need of highly sensitive assays such as the determination of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) and next generation sequencing (NGS) of immunoglobulin (IGH) gene rearrangements. Ongoing studies are examining circulating cell-free tumor DNA (cfDNA) as a sensitive measure of small amounts of residual cells. In the present study, we de- scribe and analytically validate a simplified in-house deep-sequencing method to identify and quantify residual tumor burden in MM patients from plasma samples. Methods: We retrospectively analyzed 25 MM paired tumor (n=25) and plasma samples (n=48) obtained at diagnosis and at specified time points during treatment. Genomic DNA (gDNA) and cfDNA were extracted from selected CD138+ plasma cells (PC) and from plasma (Qiagen). IGH gene rearrangements were amplified, qual- ity assessed (Agilent hsDNA kit) and sequenced on Ion Torrent PGM. Raw reads were filtered and aligned using IMGT germline database andaggregated into clonotypes. Post-processing analyses were performed using VDJtools and customized R scripts. Results: Our sequencing method successfully identified a IGH MM clonotype in 88% of tumor samples (22/25), subsequently detected in plasma of all 22 cases (me- dian 4.7% of total filtered reads). Levels of the IGH clonotype in cfDNA distinguished between groups of patients with different prognosis: pa- tients with levels >4.7% prior to therapy, had significantly shorter PFS than patients with levels<4.7% (median 268 vs 990 days; HR=3.532, P=0.04827, Log-rank). IGH cfDNA levels over the median were signif- icantly associated with higher risk of disease progression (HR=7.9, P=0.0384). cfDNA levels reflected the number of PC enumerated by MFC (r=0.7249, P<0.0001, Pearson’s correlation test). Accordingly, TTP was significantly longer (P<0.0001) for patients displaying frequencies lower than 10-5 (TTP 9±3 months, mean±SD, for frequencies>10-5 vs 15±5 months for frequencies=10-5 vs 37±4 months for frequencies<10- 5). Those patients are in CR and characterized by PC frequencies <10- 5 by MFC, and are therefore defined as MRD-negative. Conclusions: Results of this study support the clinical applicability of quantifying tumor levels by our in-house deep-sequencing of IGH gene rearrange- ments in plasma of MM patients.