Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

Lieke M J van Zogchel; Nathalie S M Lak; Nina U Gelineau; Irina Sergeeva; Ellen Stelloo; Joost Swennenhuis; Harma Feitsma; Max van Min; Erik Splinter; Margit Bleijs; Marian Groot Koerkamp; Willemijn Breunis; Michael Torsten Meister; Waleed Hassan Kholossy; Frank C P Holstege; Jan J Molenaar; Wendy W J de Leng; Janine Stutterheim; C Ellen van der Schoot; Godelieve A M Tytgat

doi:10.3389/fonc.2023.1124737

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

Front Oncol. 2023 Apr 20:13:1124737. doi: 10.3389/fonc.2023.1124737. eCollection 2023.

Authors

Lieke M J van Zogchel^{1

2}, Nathalie S M Lak^{1

2}, Nina U Gelineau^{1

2}, Irina Sergeeva³, Ellen Stelloo³, Joost Swennenhuis³, Harma Feitsma³, Max van Min³, Erik Splinter³, Margit Bleijs¹, Marian Groot Koerkamp¹, Willemijn Breunis^{1

4}, Michael Torsten Meister^{1

5}, Waleed Hassan Kholossy¹, Frank C P Holstege^{1

6}, Jan J Molenaar¹, Wendy W J de Leng⁷, Janine Stutterheim¹, C Ellen van der Schoot², Godelieve A M Tytgat¹

Affiliations

¹ Princess Máxima Center for Pediatric Oncology Research, Utrecht, Netherlands.
² Sanquin Research and Landsteiner Laboratory of the AMC- University of Amsterdam, Department of Experimental Immunohematology, Amsterdam, Netherlands.
³ Cergentis B.V., Utrecht, Netherlands.
⁴ University Children's Hospital Zürich, Zürich, Switzerland.
⁵ Oncode Institute, Utrecht, Netherlands.
⁶ Center for Molecular Medicine, University Medical Center (UMC) Utrecht and Utrecht University, Utrecht, Netherlands.
⁷ Department of Pathology, University Medical Center (UMC) Utrecht, Utrecht, Netherlands.

Abstract

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors.

Materials and methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed.

Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse.

Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

Keywords: TLA; cell-free DNA (cfDNA); liquid biopsy; neuroblastoma; paediatric cancer; patient-specific ddPCR.

Copyright © 2023 van Zogchel, Lak, Gelineau, Sergeeva, Stelloo, Swennenhuis, Feitsma, van Min, Splinter, Bleijs, Groot Koerkamp, Breunis, Meister, Kholossy, Holstege, Molenaar, de Leng, Stutterheim, van der Schoot and Tytgat.

Grants and funding

LZ was supported by Liquidhope, a TranScan-2 project by Koningin Wilhelmina Fund, KWF Kankerbestrijding TRANSCAN 8352/TRS-2018- 00000715. NL and JSt were supported by Children Cancer Free (KiKa) grant number 312. MTM received financial support from the Deutsche Forschungsgemeinschaft (#408083583). The work conducted by Cergentis has received funding from the European Union’s Horizon 2020 SME instruments program SEQURE under grant agreement No. 806446.