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

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.

Figure 1

Workflow for the development of a patient-specific assay. At primary diagnosis, tumor material is collected through biopsy or resection. The tissue is then analyzed in the regular diagnostic pipeline. This means copy number analysis through SNP array for neuroblastoma tumors, and fusion gene detection through immunohistochemistry or RT-qPCR for rhabdomyosarcoma and Ewing sarcoma. Based on the identified altered regions/copy number aberrations and fusion partner, for targeted locus amplification (TLA) or targeted locus capture (TLC) is performed on cellsor FFPE material, respectively. The breakpoint sequence(s) are then used for a patient-specific ddPCR design which is then measured on cell- free DNA from EDTA blood.

Figure 2

2D plot from the ddPCR assay for NB2049 with (A). cfDNA from the diagnostic plasma sample (total cfDNA input 25.6 ng/well) and (B). positive control with DNA from FFPE material from the primary tumor (total cfDNA input 3.3 ng/well). Blue dots; droplets positive for patient-specific breakpoint (FAM channel) Green dots; droplets positive for ACTB(HEX channel) Grey dots; droplets negative for both targets.

Figure 3

2D plot from the ddPCR assay for NB2050 with 2 patient-specific breakpoints (A). cfDNA from the diagnostic plasma sample (total cfDNA input can not be determined due to overload of the droplets) (B). positive control with DNA from FFPE material from the primary tumor (total cfDNA input 1.7 ng/well). (C) Dilution of diagnostic plasma 50 times and (D). 500 times Blue dots; droplets positive for both patient-specific breakpoint (FAM channel) Green dots; droplets positive for Actin Beta (HEX channel) Pink dots; droplets positive for breakpoint Chr 2;2 nr 1 Purple dots; droplets positive for breakpoint Chr 2;2 nr 2 (with 450 nM and 125 nM primer and probe concentrations, respectively) Orange dots; droplets positive for both breakpoints and Actin Beta Black dots; droplets positive for breakpoint nr 1 and Actin Beta Salmon-colored dots; droplets positive for breakpoint nr 2 and Actin Beta Grey dot; droplets negative for both targets.

Figure 4

Levels of patient-specific targets, reference gene ACTIN beta (ACTB) and methylated RASSF1A (RASSF1A-M) in cell-free DNA (cfDNA) from 10 neuroblastoma patients at diagnosis and during the course of the disease. Dx, diagnosis; Dx 4S, diagnosis INSS stage 4S; Dx 4, diagnosis INSS stage 4; nCt 1L, after n courses in first line therapy; nCt 2L, after courses in second line therapy; 3L, third line therapy; 1EV 3L, first evaluation third line therapy; 2EV 3L, second evaluation third line therapy; IT-0, before GD-2 immunotherapy IT-3 after 3 cycles of GD-2 immunotherapy; IT-6, after 6 cycles of GD-2 immunotherapy; MAT, myeloablative therapy and autologous stem cell transplantation; RT 2L, after radiotherapy during second line therapy; MT, maintenance treatment. PD, progressive disease; R, relapse; RD, refractory disease. Green blocks indicate first line treatment, orange blocks indicate added treatment for refractory disease, red blocks indicate treatment for progressive or relapsed disease.

Figure 5

Levels of patient-specific targets, reference gene ACTIN beta (ACTB) and methylated RASSF1A (RASSF1A-M) in cell-free DNA (cfDNA) in 2 patients with rhabdomyosarcoma (RMS026 and RMS092) and 1 patient with Ewing sarcoma (ES010) at diagnosis and during the course of the disease. Dx, diagnosis; nCt 1L, after n courses in first line therapy; M, maintenance; FUP-follow up; EVn 2L, evaluation number n during second line therapy; EOT, end of treatment; R, relapse; Start 2L, start second line therapy; R, relapse; PD, progressive disease. Green blocks indicate first line treatment, red blocks indicate treatment for progressive or relapsed disease.