Workflow evaluation for molecular characterization of single circulating tumor cells in blood samples of patients with gynecological malignancies

Janina Levermann, Norbert Hochstein, Paul Buderath, Benjamin Franken, Siegfried Hauch, Charline Bemmann, Rainer Kimmig, Sabine Kasimir-Bauer

Background: We recently presented the feasibility of single circulating tumor cell (CTC) analysis using the QIAscout single cell isolation platform (QIAGEN), followed by whole transcriptome amplification (WTA) and targeted Next Generation Sequencing (NGS). We here applied this workflow in blood samples of patients (pts) with metastatic breast cancer (MBC) and primary ovarian cancer (POC) and compared the result with a new workflow for single cell transcriptome analysis applying the QIAseq UPX 3′ Transcriptome Kit (QIAGEN) for high throughput gene expression analysis.

Methods: 5 ml blood of 7 MBC and 3 POC pts were analyzed for CTCs using the AdnaTest EMT2/StemCell SelectTM, followed single cell isolation. Adhesion of CTCs to the QIAscout array microrafts was supported by pre-treating the array with Corning® Cell-TakTM Adhesive. Single CTCs have been identified utilizing an inverted microscope, and isolated with the QIAscout single cell isolation platform. Subsequently, mRNA of single cells was amplified by WTA (REPLI-g WTA Single Cell Kit, QIAGEN) and cDNA of the cells was further analyzed by targeted NGS using a QIAseqTM Targeted RNA Panel (Human Cancer Transcriptome Panel, 395 genes). Applying the QIAseq UPX 3′ Transcriptome Kit, each single cell was tagged with an individual cell ID-sequence and each RNA molecule was simultaneously tagged with an unique molecular index (UMI) during reverse transcription. Tagging of each individual cDNA molecule enabled pooling of all samples for subsequent amplification and library preparation steps, followed by high-throughput NGS. Methods were validated in spike-in experiments using different cell lines spiked into blood of healthy donors.

Results: Using the QIAscout in combination with the WTA-workflow, we have already demonstrated an absent leukocyte contamination. However, only a very few of the detected cells in MBC pts (15%) were finally applicable for NGS. Nevertheless, analyzable CTCs expressed genes involved in angiogenesis (ITGB3), cell cycle (CCND2) and apoptosis (FASLG & SKP2). Applying the UPX 3′ Transcriptome-workflow, the number of analyzable CTCs could be markedly improved as documented in spike-in experiments as well as in blood samples of MBC and POC pts. Up to now, in the latter group, we were able to isolate a total number of 84 CTCs [median 25 CTCs (range 24-35 cells)] with 44/84 (52%) ID-sequences detected, being applicable for NGS. Experiments and detailed transcriptome profiling of CTCs are ongoing to be presented at the meeting.

Conclusion: Although both described workflows have to be further evaluated in more detail, the QIAscout single cell isolation platform allows subsequent transcriptome analysis followed by NGS to get insights into single cell heterogeneity for further therapeutic strategies.

Short title: single CTC characterization
Keywords: Breast cancer; Ovarian cance; Circulating tumor cells; single cell isolation Category: CL11-05 Biomarker: Liquid biopsies: CTCs

Janina Levermann, MSc: Janina.Levermann@uk-essen.de; Department of Gynecology and Obstetrics; University Hospital of Essen, Wissenschaftliches Labor, Ebene -1, Raum -1.08, Hufelandstraße 55, 45122 Essen, Germany

Norbert Hochstein, Ph. D: Norbert.Hochstein@qiagen.com; QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany

Paul Buderath, MD: Paul.Buderath@uk-essen.de; Department of Gynecology and Obstetrics; University Hospital of Essen, Hufelandstraße 55, 45122 Essen, Germany

Benjamin Franken, Ph. D: Benjamin.Franken@qiagen.com; QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany

Siegfried Hauch, Ph. D: Siegfried.Hauch@qiagen.com; QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany

Charline Bemmann, Charline.Bemmann@qiagen.com; QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany

Rainer Kimmig, MD: Rainer.Kimmig@uk-essen.de; Department of Gynecology and Obstetrics; University Hospital of Essen, Hufelandstraße 55, 45122 Essen, Germany

Sabine Kasimir-Bauer, Ph D: Sabine.Kasimir-bauer@uk-essen.de; Department of Gynecology and Obstetrics; University Hospital of Essen, Hufelandstraße 55, 45122 Essen, Germany

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