PB2110 MRD ANALYSIS IN MULTIPLE MYELOMA: IMMUNOGLOBULIN NGS VERSUS ASO‐PCR
Authors: Van der Straeten J. , Fostier K. , De Brouwer W. , Demanet C. , Bakkus M. H. 
Published: June 2019

Background:

MRD monitoring is a valuable tool for treatment guidance and prognostication in hematological malignancies. The real‐time quantitative allele specific oligonucleotide PCR (qASO‐PCR) is a highly standardized technique across Europe and beyond for molecular MRD detection. For multiple myeloma (MM) patients, MRD monitoring becomes also more and more important and although the qASO‐PCR is suitable in this disease, it has a lower applicability due to the lower number of targets suitable for ASO design and the presence of somatic hypermutations in the immunoglobulin (IG)‐genes. Modern techniques such as next‐generation sequencing (NGS) overcome certain limitations of this qASO‐PCR.

Aims:

The purpose of this study is to compare the IG‐NGS technique with the qASO‐PCR for MRD detection in MM.

Methods

PCR‐based NGS of the IG heavy chain (IGH) and IG kappa (IGK) genes was performed by the LymphoTrack® Dx IGH FR1/IGK Assay Panel ‐ MiSeq kits from Invivoscribe. For MRD quantification by NGS, a spike‐in of three different reference molecules (gBlocks) for each receptor was used as calibrator. The MRD data obtained by NGS were compared to the MRD data previously obtained by qASO‐PCR.

A cohort of 25 bone marrow samples of 14 MM patients was used. The samples originated from the MYVAC2‐study, a pilot study in newly‐diagnosed symptomatic MM patients ≤ 65 years achieving at least a very good partial response (VGPR) after first‐line autologous stem cell transplantation (ASCT) and for whom therapy consists of a combination of lenalidomide maintenance and 4 autologous dendritic cell vaccines against MAGE‐A3/C1. Several time points after ASCT where tested for MRD.

Results:

The analytical sensitivity of the NGS assay was comparable with the qASO‐PCR and reached 1E‐05 when 500 ng DNA was used. Dilution experiments showed a very good linearity over a 5 log range (r = 0.96).

The NGS‐MRD levels correlated well with the qASO‐PCR MRD levels in the total MM cohort (r = 0.85). The correlation was even better when the ‘positive non quantifiable’ MRD data (positive but below 1E‐04) by qASO‐PCR where eliminated (r = 0.96).

MRD monitoring by NGS was applicable for all MM patients while MRD monitoring performed by qASO‐PCR was suitable for only 8/14 patients, indicating the higher applicability of the NGS technique for MRD detection in MM. A more detailed overview of the MRD results by NGS and qASO‐PCR is given in table 1. Overall, 24% off the samples (6/25) were negative by NGS, compared to 32% (8/25) by qASO‐PCR. In the positive non‐quantifiable group by qASO‐PCR, 100% of the samples (6/6) were positive by NGS. These findings indicate that an additional 32% (8/25) of the samples could be quantitated by NGS, reflecting already the higher sensitivity/specificity of the NGS approach.

MRD negativity did result into a prolonged progression free survival benefit in the studied patient population, which validates its use as a surrogate endpoint.

Summary/Conclusion:

The PCR‐based NGS assay for MRD detection shows a good correlation with the patient‐specific qASO‐PCR. Moreover, the NGS assay is applicable in almost all MM patients while the qASO‐PCR is not, due to ASO design failure. The sensitivity of the assay is comparable with the qASO‐PCR and can only be augmented when the amount of DNA input is increased.

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