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ctDNA analysis in Lymphoma
known recurrent mutations for a particular cancer type. Those targets are then amplified and sequenced in a patient’s cfDNA sample, allowing quantification of ctDNA based on the detection of tumor-specific muta- tions, and simultaneous determination of an individual’s specific tumor mutation profile. This method can simul- taneously assay all classes of mutations, including single nucleotide variants, insertion/deletions, copy number alterations and rearrangements.18-20 The “selector” is tumor specific and requires detailed knowledge of the underlying genetic landscape of the tumor, a limitation that is currently overcome by the availability of from dozens to hundreds of genomes across all types of lym- phoma.
The clonoSEQ Assay is a diagnostic test validated and approved for measuring minimal residual disease (MRD) on genomic DNA from bone marrow samples in leukemias and myeloma.21,22 In the assay, genomic DNA is amplified by a set of locus-specific multiplex PCR using V, D and J gene primers covering all possible rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences. The amplicon library is then subjected to ultra-deep NGS. The tumor-specific clonotype is first identified in a tumor-enriched biological sample and
then tracked within the repertoire of IgH, IgK and IgL rearrangements amplified and sequenced in post-treat- ment samples. By leveraging on the advantage that the IgH, IgK and IgL rearrangements represent a stable and tumor specific fingerprint, the clonoSEQ Assay has also been applied to ctDNA quantification in lym- phomas.9,10,12 However, tracking IgH, IgK and IgL sequences has some shortcomings when applied to cfDNA, including the need for lymphoma clonotype assignment through the analysis of the tissue biopsy, limited sensitivity in low tumor burden settings, and reduced applicability because of somatic hypermutation (SHM), which is ongoing in some lymphoma types such as diffuse large B-cell lymphoma of the germinal center type and follicular lymphoma, leading to difficulties in identifying clonotypic sequences.
Overall, although methods for ctDNA identification and quantification are becoming more common, they are not yet widely used in clinical laboratories and are not, therefore, prominently featured in disease man- agement guidelines. Methodological challenges, both in molecular biology and bioinformatics analyses, must be overcome, standardized and harmonized as these meth- ods become more routinely used.
Figure 1. Schematic representation of liquid biopsy unmet needs in diagnostic, genotyping and minimal residual disease (MRD) monitoring fields with relative actions to overcome these limitations. PCNSL: primary central nervous system lymphoma; PET / CT: positron emission tomography/computed tomography; DLBCL: diffuse large B-cell leukemia; cHL: classic Hodgkin lymphoma; CAPP-seq: Cancer Personalized Profiling by Deep Sequencing.
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