A Comparison Between Next-Generation Sequencing and ASO-qPCR For Minimal Residual Disease Detection In Multiple Myeloma: The Clinical Value In ASCT Setting
Authors: Takamatsu Hiroyuki , Mura Ryoichi , Zheng Jianbiao , Moorhead Martin , Yasushi Terasaki , Takashi Yoshida , Faham Malek , Nakao Shinji 
Published: October 2019

Abstract

Background
Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We utilized a sequencing method, termed the LymphoSIGHT™ platform, which employs consensus primers and high-throughput sequencing to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. The sequencing method is quantitative at frequencies above 10-5 and the lower limit of detection is below 10-6. Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR.

Methods
We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Because myeloma cells exist patchily in bone marrow (BM), myeloma cells in PBSC autografts may reflect the whole amount of tumor in vivo. Thirty-six Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or complete response (CR) after ASCT. BM slides from 28 MM patients and fresh BM cells from 8 MM patients at diagnosis as well as autografts were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using next-generation sequencing (NGS). Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples (Faham et al, Blood 2012).

Results
MRD in autografts could be assessed 36 of 36 (100%) by NGS and 30 of 36 (83%) by ASO-qPCR. MRD in autografts was detected in 27 of 36 (75%) by NGS and 11 of 30 (37%) by ASO-qPCR (Figure 1A). Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, ASO-qPCR could not detect myeloma cells at MRD levels of 10-5 or lower. Two cases where MRD was not detected by NGS (MRDNGS(-)) and 14 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide while 7 MRDNGS(-) cases and 13 MRDNGS(+) cases were followed without post-ASCT therapy. The best post-ASCT responses were as follows: 6 (67%) mCR, 1 (11%) sCR and 2 (22%) VGPR in 9 MRDNGS(-) cases; 2 (14%) mCR, 2 (14%) sCR, 2 (14%) CR, 8 (58%) VGPR in 14 MRDNGS(+) cases with post-ASCT therapy; 2 (15%) sCR, 10 (78%) VGPR and 1 (7%) PR in 13 MRDNGS(+) cases without post-ASCT therapy. The MRDNGS(-) cases tended to show a better PFS than the MRDNGS(+) cases with post-ASCT therapy (P = 0.400) and showed a significantly better PFS than those without post-ASCT therapy (P = 0.032) (Figure 1B) although overall survival rates were comparable among the three groups. To investigate the value of sensitive detection by NGS, we compared PFS in 7 MRDNGS(-) cases (Group 1) with the 6 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 tended to show a better PFS than Group 2 (P = 0.091) (Figure 1C). This underscores the value of sensitive detection of MRD in MM.

Conclusions
A high correlation between NGS and PCR MRD results was observed, and MRD-negativity in PBSC autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR.

Disclosures:
Zheng: Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment.