Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for “personalized monitoring” of residual disease in chronic myeloid leukemia patients

Abstract

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient–specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of “personalized monitoring” of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines

“…As detailed below, the fusion of full-length FBXW7 to the promoter of FAM160A1 was also likely deleterious due to decreased expression under the new promoter ( Figure S7C ). For each of the nine predicted fusions involving at least one gene previously identified in other cancer fusions, we independently validated the novel sequence junction using PCR and Sanger sequencing and a recently developed droplet digital PCR approach ( Cumbo et al, 2018 ) ( Figures S8 and S9 ).…”

Nanopore Sequencing in Blood Diseases: A Wide Range of Opportunities

“…Nanopore technology could improve HA molecular diagnosis, particularly in those cases (about 2%) in which conventional molecular technologies fail. The nanopore ability to generate long-reads was also exploited to characterize the BCR-ABL1 genomic breakpoint in ten patients affected by CML, in order to develop an approach for the "personalized monitoring" of residual disease during followup (Cumbo et al, 2018). NS simplifies the identification of genomic junctions that are different in each patient; this procedure is difficult and expensive to accomplish with the short-read sequencing approach and not suitable for clinical practice.…”

“…NS simplifies the identification of genomic junctions that are different in each patient; this procedure is difficult and expensive to accomplish with the short-read sequencing approach and not suitable for clinical practice. The sequence thus obtained can then be used to design and test a patient-specific droplet digital PCR assay to make an absolute quantification of residual leukemic cells in patients' peripheral blood (Cumbo et al, 2018).…”

Cancer Medicine Gets Personal

“…Researchers from the University of Bari (Italy) have recently combined ddPCR with FISH and MinION, which they report created a cost-effective and reproducible method for personalized monitoring of CML that would be useful for the clinic [2]. …”