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

Lymphoid malignancies are characterized by clonal cell expansion, often identifiable by unique immunoglobulin rearrangements. Heavy (IGH) and light-chain gene usage offers diagnostic insights and enables sensitive residual disease detection via next-generation sequencing. With its adaptable throughput and variable read lengths, Oxford Nanopore thirdgeneration sequencing now holds promise for clonotyping. This study analyzed CD138+ plasma-cell DNA from eight multiple myeloma patients, comparing clonotyping performance between Nanopore sequencing, Illumina MiSeq, and Ion Torrent S5. We demonstrated clonotype consistency across platforms through Smith-Waterman local alignment of nanopore reads. The mean clonal percentage of IGH V and J gene usage in the CD138+ cells was 69% for Nanopore, 67% for S5, and 76% for MiSeq. When aligned with known clonotypes, clonal cells averaged a 91% similarity, exceeding 85%. In summary, Nanopore sequencing, with its capacity for generating millions of high-quality reads, proves effective for detecting clonal IGH rearrangements. This versatile platform offers the potential for measuring residual disease down to a sensitivity level of 10-6 at a lower cost, marking a significant advancement in clonotyping techniques.

A reference-area-free strain mapping method using precession electron diffraction data.

In this work, we developed a method using precession electron diffraction data to map the residual elastic strain at the nano-scale. The diffraction pattern of each pixel was first collected and denoised. Template matching was then applied using the center spot as the mask to identify the positions of the diffraction disks. Statistics of distances between the selected diffracted disks enable the user to make an informed decision on the reference and to generate strain maps. Strain mapping on an unstrained single crystal sapphire shows the standard deviation of strain measurement is 0.5%. With this method, we were able to successfully measure and map the residual elastic strain in VO2 on sapphire and martensite in a Ni50.3Ti29.7Hf20 shape memory alloy. This approach does not require the user to select a "strain-free area" as a reference and can work on datasets even with the crystals oriented away from zone axes. This method is expected to provide a robust and more accessible alternative means of studying the residual strain of various material systems that complements the existing algorithms for strain mapping.

Acute myeloid leukemia exhibiting clonal instability during treatment: Implications for measurable residual disease assessments.

Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45lowCD34+CD38-CD123-CD7-) and not in the putative leukemic stem cells (CD34+CD38-CD123+CD7+) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event-free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia-related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS-MRD assays.

Perspective: sensitive detection of residual lymphoproliferative disease by NGS and clonal rearrangements-how low can you go?

Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing (NGS) has provided unprecedented assay specificity, with a sensitivity matching that of polymerase chain reaction-based measurable residual disease (MRD) detection down to the 10-6 level. Although reaching 10-6 to 10-7 is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is placed in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold-in terms of both the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency to enable critical assessment of the MRD assays for clinical implementation and feasibility.