Konference: 2014 19th Congress of the European Hematology Association - účast ČR

Kategorie: Maligní lymfomy a leukémie

Téma: Acute lymphoblastic leukemia - Clinical

Číslo abstraktu: S1311

Autoři: MUDr. Michaela Kotrová; Ing. Kateřina Mužíková; MUDr. Ester Mejstříková, Ph.D.; Bc. Michaela Nováková; Mgr. Violeta Bakardjieva-Mihaylova; Mgr. Karel Fišer, Ph.D.; Mgr. Jan Stuchlý; Dr. Christianne Pott, PhD; MD Monika Brüggemann (Bruegemann), PhD; Prof.MUDr. Jan Trka, Ph.D.; prof. MUDr. Jan Starý, DrSc.; MUDr. Eva Froňková, Ph.D.


Background: MRD negativity at both day 33 and 78 is required for stratification into standard risk (SR) group in pediatric BFM protocols. Quantitative PCR (qPCR) for immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements had been standardized within the EuroMRD consortium. Next generation sequencing (NGS) of Ig/TCR repertoire is a rapidly expanding method that enables monitoring of the malignant clone, but can also show the picture of normal lymphoid cells during the treatment. Moreover, the NGS would avoid qPCR optimization for every patient, which should markedly reduce costs and handling time.

Aims: To establish the detection of multiple Ig heavy chain (IGH) rearrangements using the Ion Torrent PGM/Ion Proton platforms and to compare the NGS results with qPCR at the crucial stratification time points used in childhood BFM trials.

Methods: The sequencing libraries were created from 450 ng of bone marrow DNA mixed with 50 ng of polyclonal DNA using multiplex PCR with IGH FR3 BIOMED-2 primers. The sequencing adapters together with different multiplex identifiers allowing for parallel sequencing of different samples were introduced and sequencing was performed using the Ion Torrent 318 and Ion Proton PI chips. Bioinformatic algorithm was developed to define numbers of reads with V-D-J marker sequences selected according to their clonality in the diagnostic sample among total reads of expected size.

Results: In total 56 samples (28 x day 33, 28x day 78) from 28 patients treated according to Interim BFM 2007 protocol were sequenced with median coverage of 428,648 reads. Thirty-six samples (64%) were concordantly negative by NGS and qPCR. Five samples (9%) were negative by NGS and positive by qPCR and four samples (7%) were negative by PCR and positive by NGS (Fig.1). This would cause a shift from IR to SR in 4 patients, from SR to IR in 2 patients and from slow early response (SER) group to IR in one patient. In the 11 double-positive samples the quantitative values correlated, but the MRD levels were higher in NGS than in PCR in all but one sample (median 7x, range 1-124x). This was likely caused by the fact that NGS quantitates leukemic IGH rearrangements within the B cells only while qPCR is performed in a bulk of all mononuclear cells. Importantly, the quantitative differences would not change the stratification into treatment risk groups in any patient. After correction for percentage of CD19pos cells assessed by flow cytometry (FC) at respective time points the correlation with qPCR improved (R2=0.77).

Summary/Conclusion: In the NGS design we used, the sensitivity of NGS was comparable to qPCR, showing the most differences in the “grey zone” consisting of weakly positive samples. This caused a shift between risk groups in 7/28 (25%) patients, mostly from IR to SR. More investigated samples are needed to find out whether the differences in the risk group assignment would have clinical consequences. The NGS has a great potential for replacing antigen receptor-based qPCR in clinical decisions for ALL. At present, the main drawback of 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, NT/12397 and GACR P302/12/G101.

Keywords: Acute lymphoblastic leukemia, IgH rearrangment, MRD

Datum přednesení příspěvku: 15. 6. 2014