Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique
Authors: Kotrova Michaela , Muzikova Katerina , Mejstrikova Ester , Novakova Michaela , Bakardjieva-Mihaylova Violeta , Karel Fiser , Stuchly Jan , Pott Christiane , Bruggemann Monika , Jan Stary , Trka Jan , Fronkova Eva 
Published: October 2019

Abstract

Introduction
MRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background.
Aims
To develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints.
Methods
Two round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files.
For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution.
Results
We sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed.
Discussion
We present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks.
The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis.
Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214.

Disclosures
No relevant conflicts of interest to declare.