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

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

Rapid and reliable detection of exon rearrangements in various movement disorders genes by multiplex ligation-dependent probe amplification.

Because of the occurrence of different types of mutations, comprehensive genetic testing for Parkinson's disease (PD), dopa-responsive dystonia (DRD), and myoclonus-dystonia (M-D) should include screening for small sequence changes and for large exonic rearrangements in disease-associated genes. In diagnostic and research settings, the latter is frequently omitted or performed by laborious and expensive quantitative real-time PCR (qPCR). Our study aimed to evaluate the utility of a novel method, multiplex ligation-dependent probe amplification (MLPA), in molecular diagnostics of movement disorders. We have analyzed, by MLPA, genomic DNA from 21 patients affected with PD, DRD, or M-D, in which the presence of exon rearrangement(s) (n = 20) or of a specific point mutation (detectable by MLPA, n = 1) had been established previously by qPCR or sequencing. In parallel, we have studied, in a blinded fashion, DNA from 49 patients with an unknown mutational status. Exon rearrangements were evident in 20 samples with previously established mutations; in the 21st sample the known specific point mutation was detected. We conclude that MLPA represents a reliable method for large-scale and cost-effective gene dosage screening of various movement disorders genes. This finding reaches far beyond a simple technical advancement and has two major implications: (1) By improving the availability of comprehensive genetic testing, it supports clinicians in the establishment of a genetically defined diagnosis; (2) By enabling gene dosage testing of several genes simultaneously, it significantly facilitates the mutational analysis of large patient and control populations and thereby constitutes the prerequisite for meaningful phenotype-genotype correlations

Interleukin 6 transsignaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency

While thousands of breast cancer cells disseminate and home to bone marrow (BM) until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. Signals and mechanisms determining failure or success of disseminated cancer cells (DCCs) are largely unknown and there is no in vivo model available to study the spontaneous progression and genomic evolution from early bone marrow infiltration to manifestation of bone metastasis, as spontaneous or transgenic mouse models do not generate bone metastases. We therefore profiled DCCs from BM of breast cancer patients long before manifestation of metastasis by RNAseq to identify signals supporting survival or outgrowth of DCCs and identified IL6/PTEN/PI3K signaling as candidate pathway for DCC activation. Since early DCCs often display close-to-normal genomes we used mammary epithelial cells ex vivo isolated from reduction mammoplasties and immortalized pre-malignant breast cancer cell lines as model for functional testing in vitro. Using specific activators and inhibitors of IL6 signaling revealed that IL6 trans, but not classical signaling, regulates stemness of mammary epithelial cells. Moreover, knock-down of PTEN revealed that PI3K/PTEN pathway activation renders cells independent of IL6 trans-signaling. Interestingly, gp130 expression, a pre-requisite for IL6 trans-signaling was found to be down-regulated by bone marrow stromal and endosteal, but not vascular niche cells, and as a consequence the number of cells with stem-like ability was significantly reduced. Consistent with a bottleneck function of microenvironmental DCC control, we found PIK3CA mutations highly associated with late-stage metastatic DCCs and CTCs while generally absent in early DCCs. Our data suggest that the initial steps of metastasis formation depend on microenvironmental signals and are not cancer cell-autonomous