Background: Single-cell DNA sequencing with droplet microfluidics has enabled the characterization of genetic heterogeneity in hematologic malignancies and fresh solid tumors through the direct DNA sequencing of thousands of single cells from individual samples without the need of whole-genome amplification. The application of this technology in banked solid tumor samples has been rather limited to date, however. Here, we describe and validate a method to characterize clonal evolution of somatic mutations from frozen tumor specimens, utilizing endometrial carcinomas (ECs) as an example.

Methods: Tissue cores were obtained from frozen ECs and the extracellular matrix was enzymatically digested. A subset of extracted nuclei were used for bulk DNA extraction and targeted sequencing. The remaining nuclei were encapsulated with cell lysis buffer, and the DNA was primed and barcoded with hydrogel beads containing 317 cancer gene amplicons using a Tapestri instrument (Mission Bio). Following droplet PCR, Illumina libraries were generated and sequenced on a NovaSeq6000. Sequencing reads were trimmed and mapped to the reference genome. Barcode sequences were error-corrected and cells were detected from barcodes based on number of reads assigned to each barcode and amplicon read completeness. Genotyping was performed using GATK and the allele drop-out (ADO) rate was calculated. Non-neoplastic cells were defined by the absence of somatic mutations. Allele-specific copy number (ASCN) profiles for each tumor cell were estimated by normalizing mean reads per cell per amplicon to reads of non-neoplastic cells.

Results: We performed single-cell genotyping and ASCN profiling on two ECs with matched bulk sequencing data. For EC1, 5,004 tumor and 132 non-neoplastic cells were sequenced, for a 97% tumor purity. For EC2, 2,981 tumor and 165 non-neoplastic cells were obtained, for a 95% tumor purity. The average number of reads per amplicon per cell for EC1 and EC2 was 116 and 146 with an ADO rate of 6% and 8%, respectively. The data obtained from single-cell analysis were concordant with the matched bulk sequencing data in terms of clonal and subclonal mutations, and their respective cancer cell fraction. Single-cell sequencing further revealed the presence of loss of heterozygosity and emergence of minor subclones as late events in tumor evolution, which could not be inferred by bulk sequencing. In EC2, branch mutations displayed a spectrum consistent with microsatellite instability (MSI), reflecting its acquired MSI phenotype.

Conclusions: Here we describe a robust method to perform single-cell targeted DNA sequencing with droplet microfluidics of individual nuclei isolated from frozen tissue samples, providing the opportunity to determine the repertoire of somatic mutations at single cell resolution and to infer cancer clonal architecture and dynamics utilizing banked solid cancer tissue samples.

Citation Format: Arnaud Da Cruz Paula, Yingjie Zhu, Shirin Issa Bhaloo, Fresia Pareja, Timothy Hoang, Pier Selenica, Etta Hanlon, Thais Basili, Higinio Dopeso, Nadeem Abu-Rustum, Jorge Sergio Reis-Filho, Britta Weigelt. Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1692.