Patient-Specific Assays Based on Whole-Genome Sequencing Data to Measure Residual Disease in Children With Acute Lymphoblastic Leukemia: A Proof of Concept Study

Cecilia Arthur; Fatemah Rezayee; Nina Mogensen; Leonie Saft; Richard Rosenquist; Magnus Nordenskjöld; Arja Harila-Saari; Emma Tham; Gisela Barbany

doi:10.3389/fonc.2022.899325

Patient-Specific Assays Based on Whole-Genome Sequencing Data to Measure Residual Disease in Children With Acute Lymphoblastic Leukemia: A Proof of Concept Study

Front Oncol. 2022 Jul 5:12:899325. doi: 10.3389/fonc.2022.899325. eCollection 2022.

Authors

Cecilia Arthur^{1

2}, Fatemah Rezayee^{1

2}, Nina Mogensen^{3

4}, Leonie Saft^{5

6}, Richard Rosenquist^{1

2}, Magnus Nordenskjöld^{1

2}, Arja Harila-Saari⁷, Emma Tham^{1

2}, Gisela Barbany^{1

2}

Affiliations

¹ Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden.
² Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
³ Department of Pediatric Oncology, Karolinska University Hospital, Stockholm, Sweden.
⁴ Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.
⁵ Department of Clinical Pathology and Cancer Diagnostics, Karolinska University Hospital, Stockholm, Sweden.
⁶ Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
⁷ Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden.

Abstract

Risk-adapted treatment in acute lymphoblastic leukemia (ALL) relies on genetic information and measurable residual disease (MRD) monitoring. In this proof of concept study, DNA from diagnostic bone marrow (BM) of six children with ALL, without stratifying genetics or central nervous system (CNS) involvement, underwent whole-genome sequencing (WGS) to identify structural variants (SVs) in the leukemic blasts. Unique sequences generated by SVs were targeted with patient-specific droplet digital PCR (ddPCR) assays. Genomic DNA (gDNA) from BM and cell-free DNA (cfDNA) from plasma and cerebrospinal fluid (CSF) were analyzed longitudinally. WGS with 30× coverage enabled target identification in all cases. Limit of quantifiability (LoQ) and limit of detection (LoD) for the ddPCR assays (n = 15) were up to 10^-5 and 10^-6, respectively. All targets were readily detectable in a multiplexed ddPCR with minimal DNA input (1 ng of gDNA) at a 10^-1 dilution, and targets for half of the patients were also detectable at a 10^-2 dilution. The level of MRD in BM at end of induction and end of consolidation block 1 was in a comparable range between ddPCR and clinical routine methods for samples with detectable residual disease, although our approach consistently detected higher MRD values for patients with B-cell precursor ALL. Additionally, several samples with undetectable MRD by flow cytometry were MRD-positive by ddPCR. In plasma, the level of leukemic targets decreased in cfDNA over time following the MRD level detected in BM. cfDNA was successfully extracted from all diagnostic CSF samples (n = 6), and leukemic targets were detected in half of these. The results suggest that our approach to design molecular assays, together with ddPCR quantification, is a technically feasible option for accurate MRD quantification and that cfDNA may contribute valuable information regarding MRD and low-grade CNS involvement.

Keywords: acute lymphoblastic leukemia; diagnostic performance; disease monitoring; liquid biopsy; precision medicine; structural variation; technical feasibility; whole-genome sequencing.