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.
Highlights
B-RAF, located on the long arm of the chromosome 7 (Kamiyama et al, 1993), is one of the three RAF genes (A-RAF, B-RAF, and C-RAF) whose activation by their respective ligands (i.e., TGFalpha and EGF) induces the activation of several downstream targets, such as ERK1/2, AP1, and NFAT, with the consequent pro-proliferative and anti-apoptotic effect (Chong et al, 2003; Flockhart et al, 2009; Grimaldi et al, 2015).The B-RAF gene presents three fundamental domains: (1) CR1, the amino-terminal portion, cysteine-enriched, where BRAF interacts with RAS; (2) CR2, with regulatory activity; (3) CR3, the carboxy-terminal domain, with serine-threonine kinase activity
The purpose of the present study was to evaluate the introduction of digital droplet PCR (dd-PCR) as a molecular approach for the B-RAF V600E detection and its comparison with the QTPCR and IgH rearrangement, at diagnosis and during the follow-up of patients affected by Hairy cell leukemia (HCL)
While in literature data concerning the sensitivity of the QTPCR and the dd-PCR in HCL are few, analytic sensitivity tests have been performed at the start of the study, diluting a DNA from a HCL patient with 5% of B-RAF mutation in a pool of 5 wild-type DNAs in a half-logarithmic scale, from 1 × 10 to 1 × 10−4 dilution
Summary
The B-RAF gene presents three fundamental domains: (1) CR1, the amino-terminal portion, cysteine-enriched, where BRAF interacts with RAS; (2) CR2, with regulatory activity; (3) CR3, the carboxy-terminal domain, with serine-threonine kinase activity. The V600E mutation in exon 15 involves the CR3 domain, leading a constitutive activation of the B-RAF signal, with the consequent uncontrolled proliferative signal (Lyons et al, 2001). This aberrant kinase activity is sustained by the overexpression of some non-coding miRNA, such as miR-221 and miR-222, whose expression in HCL is strictly related to the B-RAF mutation (Ahmadzadeh et al, 2014). Mutations of B-RAF, especially the V600E, have been reported in different types of cancer (Davies et al, 2002): in the 50% of melanomas (DePeralta and Boland, 2015; El-Gamal et al, 2016) and papillary thyroid carcinomas (Jiang et al, 2016), in about 10% of colon (Tie and Desai, 2015) and ovarian cancers (Pakneshan et al, 2013), and in rare cases (1−3%) of non-small cell lung cancer (Caparica et al, 2016).
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