Identifying somatic mutations in cell‐free DNA of aggressive lymphoma patients: First cell‐free DNA results from the molecular profiling for lymphoma ( MaPLe ) study

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Introduction: Capturing the genomic landscape of a cancer located in multiple anatomical sites from a single biopsy is complicated by intertumour and intratumour heterogeneity. Recent work in solid tumours suggests that analysis of cell-free (cf) circulating tumour (ct) DNA might enable global characterisation of mutational profiles. However, cfDNA is primarily composed of DNA released by blood cells undergoing apoptosis; hence, ctDNA abundance and variant allele frequencies are very low, and comparable to the sequencing error rate, making development of reliable assays technically challenging, requiring optimisation of wet methods and bioinformatics. As part of the Molecular profiling for Lymphoma (MaPLe) study, we developed a ctDNA test to establish the relationship between variants found in lymphoma biopsy samples and those observed in the cell free DNA (cfDNA). Methods: A targeted panel of 50 genes was used to investigate cfDNA of patients from the MaPLe study with a confirmed diagnosis of diffuse large B-cell lymphoma (DLBCL). Lymphoma tissue and cfDNA from plasma were collected from 15 patients. Sequencing libraries were created using a combination of unique molecular tags (Thruplex Tag-seq) and a hybridisation and capture approach (IDT lockdown probes) and sequenced on the HiSeq2500 (Illumina). A number of different bioinformatic methods were compared: Briefly, alignment was carried out using BWA (v. 0.7.12) or Bowtie2 (v.2.2.4), deduplication was performed using Connor (v.0.5) or Curio, and variant calling was achieved using intersects of a number of different variant callers and Bayesian statistics. Results: Total cfDNA extracted from 5 to 13.5 mL plasma varied from 36 to 2259 ng, with median value of 160 ng. Despite low inputs, libraries were successfully performed from all 15 cfDNA samples. Coverage of at least 1000× was calculated to be 99% across the targeted regions. Combining dilution experiments and molecular barcoding, we have been able to accurately detect known variants at allele frequencies of 0.2%, approaching the theoretical limit of detection. Preliminary results indicate that cfDNA variants may be called at frequencies as low as 0.05% if ultradeep (100 000×) targeted sequencing is performed. Comparison of the mutational spectra identified in cfDNA and in paired biopsies will be presented, as well as a comparison of results obtained with tagged and untagged libraries and different bioinformatic approaches. Conclusion: We describe a novel highly sensitive approach for driver mutation specific detection of ctDNA in plasma from DLBCL patients. Our work in progress suggests ctDNA could be used to identify and track targetable mutations and to monitor presence of minimal residual disease. Keywords: diffuse large B-cell lymphoma (DLBCL)

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