Copy number alterations assessed at the single-cell level revealed mono- and polyclonal seeding patterns of distant metastasis in a small-cell lung cancer patient

Abstract

Dutch Cancer Society2012-RUG-5549 University of Groningen Intra-tumour heterogeneity (ITH) is a common feature of many cancers and can facilitate tumour evolution. In the present study we assessed intra-tumour copy number heterogeneity using low-coverage single-cell whole genome sequencing (scWGS) [1.Ni X. Zhuo M. Su Z. et al.Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients.Proc Natl Acad Sci U S A. 2013; 110: 21083-21088Crossref PubMed Scopus (344) Google Scholar]. We determined copy number alterations (CNAs) in single cells of two areas of the primary tumour and from mediastinal lymph node, liver and adrenal metastases of a 79-year-old female stage IV small-cell lung carcinoma (SCLC) patient (supplementary Figure S1, available at Annals of Oncology online). Copy number states in 2 Mb bins were assessed using a Hidden Markov Model in our custom developed pipeline AneuFinder [2.Van den Bos H. Spierings D.C. Taudt A.S. et al.Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer's disease neurons.Genome Biol. 2016; 17: 116Crossref PubMed Scopus (71) Google Scholar, 3.Bakker B. Taudt A. Belderbos M.E. et al.Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies.Genome Biol. 2016; 17: 115Crossref PubMed Scopus (103) Google Scholar]. Of the 586 cells analysed, 346 passed quality control and were used for further analysis (supplementary methods, available at Annals of Oncology online). Merged scWGS data of each tumour used to generate bulk CNA patterns were highly similar to those generated by array-based comparative genomic hybridization on DNA isolated from the same five tissue samples (supplementary Figure S2, available at Annals of Oncology online). Unsupervised clustering of single cell genomes for copy number similarities revealed a high degree of ITH among single cells from the primary tumour, lymph node and adrenal metastases, but a much lower degree of ITH with a distinct CNA pattern in the liver metastasis (Figure 1A). The liver CNA pattern was characterized by a disomic, a trisomic and a tetrasomic part of chromosome 11, trisomy of 18, disomy of 22 and pentasomy of Xp (Figure 1A). Previous studies have shown that metastasis development may occur from single [1.Ni X. Zhuo M. Su Z. et al.Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients.Proc Natl Acad Sci U S A. 2013; 110: 21083-21088Crossref PubMed Scopus (344) Google Scholar] or multiple subclones of the primary tumour and from one metastatic site into another [4.Gundem G. Van Loo P. Kremeyer B. et al.The evolutionary history of lethal metastatic prostate cancer.Nature. 2015; 520: 353-357Crossref PubMed Scopus (927) Google Scholar, 5.Hong M.K. Macintyre G. Wedge D.C. et al.Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer.Nat Commun. 2015; 6: 6605Crossref PubMed Scopus (260) Google Scholar]. To identify metastasis founder cells in this SCLC patient, we carried out hierarchical clustering of all sequenced primary tumour and metastasis cells. This revealed an overall intermixed pattern, especially for the primary tumours, the lymph node and adrenal metastases (Figure 1B and supplementary Figure S3, available at Annals of Oncology online). In contrast, the liver cells formed a distinct cluster that also contained two cells from primary tumour 1 (#59 and #82) and five adrenal metastasis cells (#26, #44, #74, #85 and #90), which all showed the liver-specific CNA pattern (Figure 1C). The strong association of a subset of primary tumour and adrenal metastasis single cells to the merged liver metastasis CNA data was supported by higher Pearson’s correlation coefficients to the merged liver when compared with their own merged CNA pattern (supplementary Table S1, available at Annals of Oncology online). Together, these data indicate that in this SCLC patient the liver-metastasis founder cells were present in the primary tumour as a minor clone. Hierarchical clustering of all sequenced primary tumour and metastasis cells (Figure 1B and supplementary Figure S3, available at Annals of Oncology online) and the results of the Pearson’s correlation coefficients (supplementary Figure 1D, available at Annals of Oncology online) showed close association of the liver metastasis cells, which supports the low ITH in liver metastasis. In conclusion, we found a high degree of CNA heterogeneity among cells of five distinct tumour locations in a single SCLC patient. A minority of the tumour cells of the primary tumour and the adrenal metastasis showed the dominant CNA pattern observed in liver metastasis cells. Our data suggest polyclonal seeding of the lymph node and adrenal metastases and a monoclonal seeding of the liver metastasis in this patient. We thank Nancy Halsema, Hinke G. Kazemier and Karina Hoekstra-Wakker for assistance with single-cell sequencing. We thank the operators at the central Flow Cytometry Unit (UMCG), Geert Mesander, Henk Moes and Roelof Jan van der Lei, for their assistance with sorting. We thank Kate McIntyre for editorial advice.