Comparative analysis between RQ‐PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma

Abstract

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ-PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ-PCR≥10 ). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR-negative/minor cluster region-negative/minor BCL2-negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression-free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM-; 6 PB-/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ-PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized.

Minimal residual disease (MRD) in non‐Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network

“…This is in line with previous studies showed that ddPCR and RQ exhibit a good correlation in MRD quantification in different diseases; moreover, in samples with low MRD levels, ddPCR appears able to identify a quantifiable disease more reliably than RQ and to discriminate very low MRD level from false positive samples …”

“…ddPCR appears a promising tool for MRD monitoring . Based on the dynamic nature of the method, ddPCR seems to be more accurate than RQ since (a) each sample is partitioned in thousands of droplets, representing thousands of independent PCR, and each droplet is analyzed individually; (b) small changes in fluorescence intensity are more readily detected; and (c) the ratio between target DNA molecules to PCR reagents is substantially higher .…”

“…Cells bearing the BCL2/IGH MBR translocation were quantified by RQ from each laboratory using the AbiPrism 7300 or 7900 sequence detector system (PE Applied Biosystems, Foster City, CA). The BCL2/IGH ddPCR was performed using QX100/200 (Bio‐Rad Laboratories, Hercules, CA), using the same primers employed for RQ‐PCR, as previously published …”

“…Reactions were performed using AbiPrism 7300 and 7900 sequence detector system (PE Applied Biosystems). The IGH ddPCR experiments were performed using QX100/200 (Bio‐Rad Laboratories), as published, using the same RQ‐PCR primers.…”

New Molecular Technologies for Minimal Residual Disease Evaluation in B-Cell Lymphoid Malignancies

“…So far, only a few studies, afterwards described, report dPCR use for MRD monitoring in lymphoprolipherative disorders [ 23 , 42 , 43 , 44 , 45 , 46 , 47 ]. Actually, more progresses have been achieved by dPCR for MRD evaluation in other hematological malignancies.…”