Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS

Abstract

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.

Kappa and lambda immunohistochemistry and in situ hybridization in the evaluation of atypical cutaneous lymphoid infiltrates

“…77 In summary, these studies help to support a diagnosis of lymphoma, but may be limited by insufficient or low-quality DNA, variability in the annealing of primer sets to the target sequences, somatic hypermutation, clonal heterogeneity, or variability/difficulty in the interpretation of monoclonal peaks. [77][78][79][80] Consequently, monoclonal gene rearrangements may be found in lesions that do not meet other clinical or histopathologic features of a lymphoma, 5,17,27,40,64 and neoplastic B-cell and plasma cell populations may not be recognized as monoclonal. These studies may miss clonal B-cell populations in as many as 50% of cases.…”

“…Next-generation sequencing (NGS) is emerging as a tool for the detection of clonal immunoglobulin light chain gene rearrangement studies. 79 Although more studies are needed, this technique may be more sensitive at recognizing a monoclonal B-cell population, even in low-density, poor quality samples. In addition, NGS can be used to simultaneously assess multiple proteins important for diagnosis or prognosis.…”

Capture-Based Next-Generation Sequencing Improves the Identification of Immunoglobulin/T-Cell Receptor Clonal Markers and Gene Mutations in Adult Acute Lymphoblastic Leukemia Patients Lacking Molecular Probes

“…Therefore, MRD stratification using the gold standard ASO-qPCR approach is nowadays feasible in about 95% of cases [ 12 ]. NGS technology has been demonstrated to be a powerful tool for the identification of clonal rearrangements at disease presentation, resulting in being less laborious and easier to perform than standard techniques [ 14 , 15 , 16 ]. Indeed, NGS proved faster compared to multistep standard workflow, allowing an efficient patient-specific probe design for timely MRD assessment, based on NGS-derived sequences.…”

“…New high-throughput technologies have become available and are being explored in order to overcome the limits of the conventional and standardized MRD assessment [ 13 ]. A next-generation sequencing (NGS) approach, similarly based on rearrangement amplification by PCR, has been described to identify clonal markers at diagnosis [ 14 , 15 , 16 ] and to monitor MRD in lymphoid malignancies [ 17 , 18 , 19 , 20 , 21 ]. All in all, NGS-based Ig/TCR marker screening greatly improved MRD monitoring, being less time consuming, and highlighted the polyclonal false-positive scenario.…”

Monitoring minimal residual disease in the bone marrow using next generation sequencing

“…We have implemented LymphoTrack as standard of care in the pathology laboratory at Memorial Sloan Kettering Cancer Center and have excellent experiences using both assays (24,28,29,31). As an non-commercial alternative, a new set of NGS-based MRD assays were recently published by the EuroClonality/BIOMED-2 consortium (32,33). Although the research efforts of the BIOMED-2 consortium is primarily acute lymphoblastic leukemia, their assays have an excellent track-record, and the previous version has been applied successfully to multiple myeloma (34).…”