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A novel workflow for isolation and multi-omic profiling of DCCs derived from cerebrospinal fluid of patients with pediatric brain cancer

: Czyz, Zbigniew T.; Feliciello, Giancarlo; Guzvic, Miodrag; Schamberger, Thomas; Grunewald, Sandra; Corbacioglu, Selim; Riemenschneider, Markus J.; Polzer, Bernhard; Klein, Christoph A.


Cancer research 79 (2019), No.13, Supplement, Abstract LB-239
ISSN: 0008-5472
ISSN: 1538-7445
ISSN: 0099-7013
ISSN: 0099-7374
American Association for Cancer Research (AACR Annual Meeting) <2019, Atlanta/Ga.>
Fraunhofer ITEM ()

Background: Clinical management of cancers of the central nevus system (CNS) is very challenging as they often exhibit low responsiveness to radiation and chemotherapy resulting in overall poor survival. Moreover, analysis of mechanisms driving cancer progression and selection of targeted therapies in CNS tumors is hindered by the limited availability to tumor tissues accessible only though surgical biopsies. A potential source of cancer material in patients with CNS is cerebrospinal fluid (CSF). Analysis of CSF-derived disseminated cancer cells (csfDCCs) holds a promise for improvement of diagnostics and monitoring of CNS tumors. For this reason, we developed a novel workflow allowing detection, isolation and multi-omic analysis of csfDCCs.
Methods: In a proof of concept study a new workflow was used to analyze CSF samples from two patients with medulloblastoma and pineoblastoma. CSF-derived cells were stained for CD45 to allow identification of infiltrating immune cells. Putative csfDCCs (CD45-negative) and control cells (CD45-positive) were subjected to a multi-omic workflow allowing parallel sequencing of genomes and transcriptomes of the same cells. Single-cell mRNA was physically separated from DNA, amplified by means of whole transcriptome amplification (Ampli1 WTA) and analyzed using endpoint PCR and a proprietary single-cell RNA-Seq approach. In parallel, DNA was subjected to whole genome amplification (Ampli1 WGA) and analyzed for the presence of copy number variations as well as point mutations (Ampli1 LowPass Kit and targeted sequencing of actionable hot-spots).
Results: We analyzed seven CNS-derived single cells and five cell clusters from the medulloblastoma patient and further eight cell clusters and eight single cells obtained from the pineoblastoma patient. Transcriptome analysis revealed that expression of neural lineage markers (e.g. CD133, SYP, OTX2, MSI1, MAP2, NEUROG1 and NEUROD1) is present almost exclusively in CD45-negative cells. Only two samples collected from medulloblastoma patient co-expressed CD45 and GFAP. However, DNA analysis revealed that CD45-positive and CD45/GFAP double-positive samples showed non-aberrant genomic profiles, thus these cells were classified as non-malignant. In contrast, all CD45-negative cells harbored genetic alterations confirming their malignant origin.
Conclusion: Our proof of concept study shows a novel workflow allowing identification, isolation as well as parallel genome and transcriptome analysis csfDCCs of patients with pediatric CNS tumors.