Nanopore sequencing of clonal IGH rearrangements in cell-free DNA as a biomarker for acute lymphoblastic leukemia

Abstract

BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL.MethodsWe present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status.ResultsQuantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample.ConclusionsThe Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

SWIGH-SCORE: A translational light-weight approach in computational detection of rearranged immunoglobulin heavy chain to be used in monoclonal lymphoproliferative disorders

“…A low error rate is crucial when targeting low MRD levels, such as 10 −4 –10 −6 . While there have been attempts to use nanopore technology for MRD detection, such as the study by Sampathi et al [ 30 ], small sample sizes limit these efforts and lack comparisons with more accurate sequencing platforms. However, the technology seems to be continuously refined, such as the currently available R10 flow cells and chemistry, suggesting that the sequencers might be an option for MRD assessment in the future.…”

Beyond assembly: the increasing flexibility of single-molecule sequencing technology

“… 128 , 129 ). As reported in both published and preprint articles, cfDNA has been sequenced with PacBio and ONT to detect fetal DNA in maternal blood 130 , 131 and assay circulating tumour DNA 132 – 135 . The ability to measure native CpG methylation and patterns from fragment ends (known as ‘fragmentomics’ 129 ) has been used to classify placental and maternal DNA 130 , show that tumour-derived DNA had lower methylation than non-tumour-derived DNA 132 , estimate tissue-of-origin and cell-type proportions (Fig.…”

The potential of 3rd‐generation nanopore sequencing for B‐cell clonotyping in lymphoproliferative disorders

“…Thus, our findings remain to be evaluated in frank MRD settings. However, it has recently been demonstrated in a preliminary and interesting study by Sampathi et al that this is at least possible using cell-free DNA from acute lymphoblastic leukemia [13].…”