The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia

Abstract

Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by “classic” HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to “classic” quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.

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

“…Although in a limited number of samples, it is worth to notice the absence of interlaboratory discordance when ddPCR was applied to brd samples. This is in line with previous studies showed that ddPCR and RQ exhibit a good correlation in MRD quantification in different diseases [16][17][18]27,28 ; 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. 32 Next-generation sequencing (NGS) has also been shown to represent an excellent tool for MRD detection in precursor and mature B-cell tumors.…”

“…ddPCR appears a promising tool for MRD monitoring. [16][17][18]27,28 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. [29][30][31] Since February 2015, we have included also ddPCR in the QC rounds.…”

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.…”

“…In a recent study, Guerrini F. et al [ 46 ] used droplet dPCR for the detection of B-RAF V600E mutation in HCL; MRD detection by this technique was significantly correlated to clinical status. Moreover, in this study, the authors compared the two quantitative methods showing a sensitivity of droplet dPCR more than half a log higher than qPCR, similar to the sensitivity levels reported for MYD88 L265P mutation detection; in fact, in WM patients, droplet dPCR reached a sensitivity of 5.0 × 10 −5 , compared to the 1.0 × 10 −3 achievable by ASO-PCR [ 53 ].…”

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

“…MRD is the major cause of relapse in cancers [29]. Recently, ddPCR has been shown to have promise in MRD monitoring where assays with high sensitivity are [30] proposed that ddPCR would be applicable in the monitoring of MRD and follow-up of patients with Hairy cell leukemia, as it showed higher sensitivity in detecting B-RAF mutation compared with qPCR (0.005% vs. 0.025%). Moreover, the detection limit in B-RAF mutations was 0.0005% when ddPCR was combined with whole genome amplification [31].…”