Whole genome sequencing in pediatric ALL - a tool for understanding chromosomal aberrations and improving diagnostic procedures

Whole genome sequencing is a revolutionary technology that has changed the field of genomics. By providing unprecedented base pair resolution, WGS allows for precise identification of disease causing genetic abnormalities. While cytogenetic and molecular methods are well established in clinical practice, the application of WGS in cancer diagnostics remains at the developmental stage. Additionally, detecting complex genomic events currently requires a multi- modal approach, as each method is targeted and has limited resolution. This reliance on multiple techniques often leads to cumbersome workflows and requires input from various experts with specialized knowledge.

One of the diseases that requires genetic characterization is acute lymphoblastic leukemia (ALL). This disease is known to be genetically heterogeneous, with structural genomic changes playing a central role in leukemogenesis. Currently, ALL is divided into genetic subgroups, each associated with different outcomes. Detecting and identifying the genetic aberrations driving disease progression often requires the use of multiple methods. Therefore, having a single method capable of detecting all genetic aberrations would be highly beneficial.

In this thesis, the efforts to assess the value of WGS as a stand-alone method in clinical diagnostics are described. The aim is not only to detect the genetic aberrations described in the treatment protocols, but also to identify emerging genetic subgroups and novel gene fusions that have diagnostic significance or can be used for targeted therapy.

In Paper I WGS was applied to a pediatric B-cell ALL (B-ALL) cohort to investigate its feasibility to detect clinically relevant genetic aberrations. Two sequencing depths (30x versus 90x coverages) and two analysis approaches (leukemia-only vs leukemia/normal) were tested, and the results were comprehensively compared to findings from standard of care (SoC) methods. Based on our analysis, sequencing leukemia samples with 30x coverage was sufficient to detect all obligatory aberrations. Additionally, almost all patients without stratifying genetic markers could be allocated to one of the emerging subgroups. The paper demonstrates the potential of WGS in the diagnostic setting of ALL .

In Paper II we applied WGS to genetically characterize a case of pediatric T-cell ALL (T-ALL) that did not respond to standard therapy. WGS detected a novel JAK2:CCDC88C gene fusion, resulting in a chimeric transcript. This novel transcript, containing the tyrosine kinase domain of JAK2, produces a chimeric protein expected to respond to a JAK2 inhibitor. This case report shows how WGS can be used to find personalized treatment options for leukemia patients.

In Paper III we present a proof-of-concept study describing the use of patient- specific genetic markers detected by WGS to monitor disease. This is performed by designing sensitive quantitative assays targeting these genetic markers. WGS was applied to detect structural variants (SVs) and other relevant leukemic genetic alterations resulting in unique sequences in six pediatric ALL cases. We then used this data to identify patient-specific targets and designed droplet digital PCR (ddPCR) assays to enable the absolute quantification of these targets. Subsequently, the technical feasibility of these patient-specific targets was assessed for monitoring measurable residual disease (MRD) in genomic DNA and cell-free tumor DNA. The results showed that the sequences provided by WGS enabled us to identify patient-specific targets that could be used for sensitive and specific detection of leukemic cells.

Manuscript IV explores the complexity of a specific ALL subtype. We applied WGS and whole transcriptome sequencing (WTS) to perform a detailed characterization of the genomic and transcriptomic profiles of Philadelphia- positive ALL (Ph+ALL). We have integrated the findings from SoC diagnostics and MRD information to comprehensively investigate the features of Ph+ALL and potential for refining diagnostics for these patients.

Together, these studies explored the potential of WGS in clinical diagnostics and enhanced the understanding of genetic aberrations in pediatric ALL.

List of scientific papers

I. Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia. F. Rezayee, J. Eisfeldt, A. Skaftason, I. Öfverholm, S. Sayyab, A. C. Syvänen, K. Maqbool, H. Lilljebjörn, B. Johansson, L. Olsson- Arvidsson, C. Orsmark Pietras, A. Staffas, L. Palmqvist, T. Fioretos, L. Cavelier, L. Fogelstrand, J. Nordlund, V. Wirta, R. Rosenquist, and G. Barbany. Front Oncol 2023, 13:1217712. https://doi.org/10.3389/fonc.2023.1217712

II. Case Report: Whole genome sequencing identifies CCDC88C as a novel JAK2 fusion partner in pediatric T-cell acute lymphoblastic leukemia. A. Krstic, F. Rezayee, L. Saft, A. Hammarsjö, P. Svenberg, and G. Barbany. Front. Pediatr. 2023, 10:1082986. https://doi.org/10.3389/fped.2022.1082986

III. Patient-specific assays based on whole-genome sequencing data to measure residual disease in children with acute lymphoblastic leukemia: a proof of concept study. C. Arthurt; F. Rezayee; N. Mogensen; L. Saft; R. Rosenquist; M. Nordenskjold; A. Harila-Saari; E. Tham; G. Barbany Front Oncol 2022, 12, 899325. https://doi.org/10.3389/fonc.2022.899325

IV. Unveiling the complexity of Philadelphia-positive acute lymphoblastic leukemia through the integration of genomic, phenotypic, and measurable residual disease data. F. Rezayee, J. Eisfeldt, A. Krstic, P. Svenberg, J. Joelsson, K. Belander Strålin, H. Vogt, S. Deneberg, S. Volanthen, L. Saft, R. Rosenquist, and G. Barbany. [Manuscript]