Next-Generation Sequencing and Real-Time Quantitative PCR For Quantification Of Low-Level Minimal Residual Disease In Acute Lymphoblastic Leukemia Of Adults
Authors: Brüggemann Monika , Ritgen Matthias , Ladetto Marco , Pepin Francois , Trautmann Heiko , Carlton Victoria , Hoelzer Dieter , Gökbuget Nicola , Kneba Michael , Faham Malek , Pott Christiane 
Published: October 2019

Abstract

Background
Detection of minimal residual disease (MRD) in adult ALL is an independent risk parameter and early indicator of an impending relapse. MRD diagnostics have been incorporated in many ALL treatment protocols as a tool for pre-emptive treatment or risk group stratification. Currently, real-time quantitative (RQ)-PCR of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements is regarded as the most sensitive and standardized method to quantify MRD in BCR-ABL negative ALL. However, IG/TR oligoclonality and clonal evolution may lead to false negative results, while sensitivity varies depending on background amplification and is generally limited to 1E-05. Next-Generation Sequencing (NGS) of IG/TR gene rearrangements might overcome these limitations and potentially provide further increases in sensitivity, specificity, accuracy and reproducibility. We have performed a comparison of the two approaches on 130 diagnostic (DG), relapse (REL) and remission (FU) samples of 14 adult patients (pts) with B-cell precursor ALL. To test the NGS sensitivity, we partly selected follow-up samples for (low-level) RQ-PCR MRD positivity or a high pre-test probability of residual disease (either due to a later relapse or MRD detection in temporal relation to the analyzed sample).

Patients and Methods
130 samples (97 bone marrow, 31 peripheral blood, 2 stem cell apheresis; 15 DG, 107 FU, 8 REL samples) were analyzed from 14 pts with B-cell precursor ALL. IG/TR-based RQ-PCR was carried out within routine diagnostics in the context of prospective clinical trials [Brüggemann et al., Blood 2006], according to the EuroMRD criteria [van der Velden et al., Leukemia 2007] at the MRD reference laboratory in Kiel. NGS was performed at the Sequenta facilities in South San Francisco. Using universal primer sets, IGH, IGK, TRB, TRG, and TRD variable, (diversity), and joining gene segments were amplified from DG and REL DNA. PCR products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in DG samples. The presence of the tumor-specific clonotype was then quantitated in FU samples. A quantitative and standardized measure of clone level among all leukocytes was determined using internal reference DNA. Comparability of MRD results by RQ-PCR and NGS was assessed using bivariate correlations between methods analysis (software R 3.0.1 with cor.test).

Results
Sanger sequencing of multiplex PCR detected 60 clonal IGH, IGK, TRB, TRG and/or TRD gene rearrangements at diagnosis (1-6/patient), NGS identified 57 index sequences (1-6/patient). Using NGS, relapse samples were analyzed for clonal evolution: 16/19 index rearrangements of relapsing pts were stable at relapse while 3/19 rearrangements (2 IGH VH-JH and 1 IGK) were lost at relapse.
Based on Sanger sequence information of clonal rearrangements 1-3 RQ-PCR assays/patient were established while NGS focused on 1-4 clonal markers/patient.
MRD was quantified in 107 FU in remission using both tools. A mean of 223,086±48,030 cells were analyzed per target using RQ-PCR, whereas NGS used 575,776±304,653 cells. An excellent concordance between the two methods was observed (p<0.0001, r2 = 0.974) with 46/107 samples being MRD positive and 45/107 MRD negative with both tools. Sequencing detected MRD in 13 samples that were negative by RQ-PCR whereas RQ-PCR revealed MRD in 3 NGS negative samples. All discrepancies affected samples with low MRD levels (below quantitative range (RQ-PCR) and levels below 8E-06 (NGS)) being consistent with Poisson sampling. In addition, both methods showed quantitative and qualitative inter-target differences with 11/49 RQ-PCR and 32/59 NGS positive samples being negative for at least one target illustrating the importance of focusing on more than one index sequence for RQ-PCR and NGS.

Conclusions
Significant concordance was observed between NGS and RQ-PCR, with NGS having similar or slightly better sensitivity compared to standardized RQ-PCR. This can be partly attributed to the higher number of cells analyzed by NGS and points out that sensitivity advantages afforded by the NGS platform may be achieved by higher input cell amounts. Prospective analyses of unselected cases must be performed to verify the clinical impact of low level NGS-based MRD detection.

Disclosures:
Pepin: Sequenta Inc.: Employment, Stockholder Other. Carlton:Sequenta Inc.: Employment, Stockholder Other. Faham:Sequenta Inc.: Employment, Stockholder Other.