Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Abstract

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

Keywords: cancer diagnostics; personalized therapy.

Fig. 1.

Primary and metastatic tumors and CTC from patient 1 who experienced a phenotypic transition from ADC to SCLC. The H&E staining and immunohistochemical staining for synaptophysin (SYN) show a typical ADC in the lung (Left) and a typical SCLC in the liver (Right) (image magnification: 200x). The CT images show the preoperative primary tumor in the lower lobe of the right lung (yellow arrow) and the metastatic posttreatment tumor in the right lobe of the liver (blue arrow). In the Center, a circulating tumor cell is identified by positive staining for DAPI and cytokeratin (Cyto), and negative staining for CD45. As a control, a leukocyte is also shown (DAPI+, Cyto−, CD45+).

Fig. 2.

Detection of somatic mutations (SNVs and INDELs) in CTCs and primary/metastatic tumors of patient 1. (A) Nonsynonymous heterozygous (hetero.) and homozygous (homo.) mutations in the lung primary (Pri.) tumor, eight CTCs, and the liver metastatic (Meta.) tumor. The blank region represents no sequence coverage. The mutated genes are listed in the right column. (B) Venn diagram of the nonsynonymous SNVs and INDELs among the lung primary tumor, CTCs, and the liver metastatic tumor of patient 1. (C) Venn diagram of the nonsynonymous SNVs and INDELs that are reported in the COSMIC database.

Fig. 3.

CNVs in CTCs from six patients with ADC and one patient with a mixture of ADC and SCLC. (A) All eight CTCs in patient 1 with reproducible CNV patterns. The copy numbers were segmented (blue and red lines) with HMM. (B) CNV patterns of CTCs from six ADC patients (patients 1–6) and a patient with a mixture of ADC and SCLC (patient 7). Patient 1 experienced a phenotypic transition from ADC in the lung to SCLC in the liver. Patient 7 was diagnosed as a mixture of ADC and SCLC in the lung. In each patient, sequencing data from all CTCs were combined for CNV analyses. (C) Clustering analyses of CTCs based on the CNVs. CTCs from patients 1 and 7 were separated from CTCs from other five ADC patients according to the analyses.

Fig. 4.

CNVs and SNVs/INDELs of SCLC. (A) Four SCLC patients (patients 8–11) with heterogeneities in their CNV patterns. In each patient, sequencing data from all CTCs were combined for CNV analyses. (B) Fraction of mutation frequency of 152 SNVs/INDELs across CTCs before (blue), and during the first-line (red) and second-line (green) chemotherapy (chemo.) in patient 8. (C) CTCs from patient 8 with constant CNVs at different therapeutic stages. Four CTCs from each stage were shown in this plot (see SI Appendix, Figs. S12 and S17, for CNVs of all CTCs from this patient).