Chromosome instability (CIN) is a hallmark of cancer, acting by boosting genetic alterations responsible of tumorigenesis, progression and heterogeneity. Whole-genome sequencing (WGS) protocols are established methods for studying copy-number alterations (CNA) in single-cells, following a necessary whole-genome amplification (WGA) step. We previously presented a method for single-cell CNA profiling of CTCs based on shallow WGS of LM-PCR based WGA products. Here for the first time, we show that the same method may be employed even on single FFPE cells, overcoming the challenges of DNA degradation and damage linked to this type of samples.
Two 50μm thick FFPE sections from pancreas ductal adenocarcinoma with <20% tumor content were processed following DEPArray™ sample preparation protocol, including cell dissociation and staining with vimentin, keratin and DAPI, and DNA quality was assessed using the DEPArray FFPE QC kit (QC=0.7). Cell pools (n=15, range=6-213 cells) and single-cells (n=11) from pure tumor and stromal populations were recovered in separate tubes and lysed. A pool of 86 tumor cells and a pool of 213 stromal cells were used for low-pass WGS on MiSeq, obtaining ≈2M paired-end reads on average. By contrast, other cell pools and single cells were firstly whole-genome amplified using the Ampli1™ WGA kit and then sequenced at shallow coverage using Ampli1 LowPass kit and IonTorrent PGM, with ≈500k single-end reads as mean throughput per library. The genome integrity (GI) of WGA samples was evaluated by Ampli1 QC, a PCR-assay based on 4 amplicons of different lengths.
The purity of DEPArray sorting was confirmed by the large number of chromosome alterations in sorted tumor and the parallel lack of gains and losses in sorted stromal pools and single-cells. A titration test was conducted measuring the consistency of CNA profiles of tumor WGA products starting from different number of cells, ranging from 117 down to single cells. For the purpose, we employed a Receiver Operating Characteristic (ROC) curve using the non-amplified tumor population as reference. Results showed an excellent performance level with a mean Area Under Curve (AUC) equals to 0.93. Interestingly, lower AUCs (0.87) were observed for single-cells, due to some level of inter-cell heterogeneity. Moreover, the proposed low-pass WGS method demonstrated a high resiliency to DNA degradation as quality of CNA profiles, measured by Derivative Log Ratio Spread (DLRS), only showed a weak correlation with GI level, with high-quality CNA profiles obtained also with the lowest GI value.
Presented approach for copy-number profiling of tumor single-cells isolated by DEPArray digital sorter and processed with Ampli1 workflow has proven to be a reliable and valuable application for the molecular characterization of tumor clones in degraded samples as FFPE tissues.
Citation Format: Alberto Ferrarini, Genny Buson, Chiara Bolognesi, Claudio Forcato, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2017-3945