Digital PCR for quantification of recurrent and potentially actionable somatic mutations in circulating free DNA from patients with diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments.

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

“…To investigate the feasibility of ddPCR for MYD88 p.(L265P) detection, it was first analyzed whether results of ddPCR on FFPE material were comparable to those of former NGS analysis. As shown in Table , the results of ddPCR and NGS matched perfectly, which corresponds to previous studies that investigated correlation of NGS and ddPCR for MYD88 p.(L265P) detection or proved applicability of ddPCR for MYD88 p.(L265P) detection for several non‐Hodgkin lymphomas on FFPE material, as well as on fresh tumor tissue and in liquid biopsies (blood) …”

“…Considering the complication risk of a brain biopsy, mutation detection in CSF is preferred. 20,21 In addition, we have shown that ddPCR is a highly reliable and sensitive method for detection of MYD88 p.(L265P) in CSF ( Notwithstanding the previously mentioned advantages of ddPCR, one must realize that NGS is becoming more and more standard procedure for analyzing lymphomas. Also, NGS is evolving rapidly and is increasingly able to analyze small DNA concentrations.…”

Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma

“…22 Notably, Oki et al 23 successfully detected tumor-specific immunoglobulin gene segments in blood, indicating that the principle of liquid biopsy may also be relevant for cHL patients. We recently designed a highly sensitive and specific probe-based digital polymerase chain reaction (dPCR) assay for the detection of XPO1 E571K somatic mutations in plasma cfDNA 24 that could be used as a tool to detect minimal residual disease (MRD).…”

Assessment of circulating tumor DNA in pediatric solid tumors: The promise of liquid biopsies

“…28 Beyond the studies examining ctDNA in solid tumors, there is a growing body of literature evaluating ctDNA in adult patients with hematologic malignancies. [29][30][31][32][33][34][35][36] Although ctDNA has been evaluated in the peripheral blood of children with leukemia, these assays lack the sensitivity of modern minimal residual disease assays (MRD). 37,38 In CNS tumors, detection of ctDNA in the peripheral blood is possible, but sensitivity appears to be a challenge in these patients.…”