Introduction

Several methods are currently available for Circulating Tumor Cells (CTCs) enrichment. Many studies compared CTC counts between FDA-approved CellSearch® system (based on EpCAM enrichment) and size-based selection methods. However, little is known about genomic differences across CTCs obtained from different platforms. For the first time, we report here a genomic characterization of copy-number and sequence variants in single CTCs enriched with different methods from the same patient.

Methods

Peripheral blood (PB) and FFPE tissue from a pelvis bone metastasis were collected from an advanced lung adenocarcinoma patient, treated with cisplatin-pemetrexed-necitumumab as first line therapy and with carboplatin-gentamicin as second-line. Two PB samples were collected at the same time: one was enriched with CellSearch® System, and one with Parsortix PR1 (size-based selection), followed by Cytokeratin-APC, CD45-PerCp and DAPI staining.

Both samples were analyzed with DEPArray™ system and single CTCs along with single WBCs as controls, were isolated.

Collected cells were whole genome amplified with Ampli1™ WGA kit and their Genome Integrity Index (GII) was assessed. On IonTorrent™ PGM Platform, we profiled Genome-wide copy-number alterations (CNA) by low-pass whole-genome sequencing with Ampli1™ LowPass Kit, and analyzed cancer-gene sequence variants with Ampli1™ CHP Custom Beta targeted panel.

FFPE tissue was disaggregated down to single-cell suspension and labelled with Keratin and Vimentin to distinguish tumor from stromal cells. Aliquots of pure cells were recovered with DEPArray™ system; the analysis of CNVs and mutations on tissue-derived samples is in progress.

Results

Single CTCs (CK+, CD45-,DAPI), showing GII≥3, either isolated from CellSearch®-enriched or PR1-enriched blood, along with WBC, were selected for NGS.

Ampli1™ LowPass Kit data showed several aberrations confirming tumor origin of all CTCs, while WBCs (n = 6) produced balanced profiles. Unsupervised hierarchical clustering segregated PR1-derived (n = 6) from CellSearch®-derived (n = 4) single CTCs in two separate branches. PR1-derived CTCs were very similar to each other, whereas CellSearch®-derived CTCs were more heterogeneous, although certain aberrations were common to all CTCs across platforms. At the sequence level, the targeted panel revealed somatic mutations shared by all CTCs (FLT3), a PTPN11 subclonal mutation (in 3/6 PR1 and 1/4 CellSearch), and other private mutations in single CTCs.

Discussion

The combination of low-pass whole-genome sequencing and targeted sequencing on single-CTCs sorted from PB enriched with different platforms reveals genetic similarities and diversities.

Integration of results with genetic analysis of pure tumor cells isolated from the tumor tissue, will provide further insight of tumor diversity in different compartments.

Citation Format: Francesca Fontana, Francesco Gelsomino, Claudio Forcato, Mario Terracciano, Chiara Bolognesi, Annalisa Altimari, Genny Buson, Chiara Mangano, Paola Tononi, Francesco Bacchi, Gianni Medoro, Michelangelo Fiorentino, Nicolò Manaresi, Michele Tognetto, Andrea Ardizzoni. Digital sorting and single-cell genomic profile comparison of lung adenocarcinoma CTCs between EpCAM and size-based enrichment methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2253.