The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. Circulating and disseminated tumor cells (CTCs and DTCs) are cancer cells that dissociate from primary and metastatic cancer sites and enter the circulation with potential to seed distant metastases. CTCs can be enriched or isolated from a simple blood liquid biopsy. Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response.
Studies (Author, Year) |
CTC Isolation | CTC Recovery | WGA Kits | Downstream Molecular Analysis | CTCs+ Patients Analyzed | CTC Nr Analyzed for WGA | Main Findings in Genetic Mutations and Alterations |
---|---|---|---|---|---|---|---|
mBC or HER2- mBC | |||||||
Babayan, A. et al., 2013 [20][4] | Density gradient | Micromanipulator TransferMan NK2 |
PicoPlex | Multiplex PCR | 4 | 8 single CTCs | ESR1 mutations in exons 4, 6 and 8 were not found |
De Luca, F. et al., 2016 [68][14] | CellSearch | DEPArray | Ampli1 | NGS (Ion AmpliSeq Cancer Hotspot panel v2) | 4 | 3–5 single CTCs per patient | 51 sequence variants in 25 genes were found, including somatic mutations in TP53 (8 mutations) and PDGFRA (3 mutations). High intra- and inter-patient heterogeneity, discordance in mutational status between CTCs and primary tissue |
Gasch, C. et al., 2016 [64][13] | CellSearch | Micromanipulator TransferMan NK2 | GenomiPhi, Ampli1 | Sanger sequencing, PCR |
33 | 114 single CTCs | PIK3CA mutations in exon 9 and 20 |
Kaur, P. et al., 2020 [70][15] | Microfluidic ANGLE Parsortix |
NA | REPLI-g | WES (SNVs, CNAs and SVs) | 5 | 5 CTCs and 5 WBCs | Elevated C>T mutational signature in patient samples. Low VAFs for somatic variants in CTCs compared to metastasis, complex rearrangement patterns were observed, high discordance between paired samples, marked heterogeneity of somatic landscape |
Li, S. et al., 2020 [59][2] | CellCollector | CellCollector | REPLI-g | NGS (HiSeq X-Ten Illumina) | 17 | 0–15 CTCs | Different metastatic sites have their own corresponding high-frequency mutation genes |
Neumann, M. H. et al., 2016 [65][16] | CellSearch | CellCelector | Ampli1 | For library preparation, the multiplex PCR-based Ion Torrent AmpliSeqTM technology with Ampli1 CHPCustom Beta panel |
2 | 7 single CTCs | Functional PIK3CA SNP (G to A, E545K) was detected in CTCs of patient 1 but not in CTCs of patient 2 |
Neves, R. P. et al., 2014 [63][12] | CellSearch | FACS | Ampli1 | aCGH (CNAs), qPCR | 30 | 192 single CTCs | 72.9% WGA success rate, 46.2% of WGA products show CCND1 amplification, mutations in PIK3CA exon 20 in c.3140 were found in CTCs (2/12 analyzed patients), TP53 mutations in exons 5, 7 and 8 were not found |
Paolillo, C. et al., 2017 [25][5] | CellSearch | DEPArray | MALBAC | Sanger sequencing | 3 | 40 single CTCs and 12 WBCs | ESR1 mutations (Y537S and T570I) were identified |
Pestrin, M. et al., 2014 [26][10] | CellSearch | DEPArray | Ampli1 | Sanger sequencing (hotspot regions in PIK3CA exon 9, 20) | 18 | 115 single CTCs | 33% of patients had an identified PI3KCA mutation. Six different mutations in the PI3KCA gene, such as c.3140A>G, c.1633G>A, c.1624G>A, c.1624G>A, etc., were identified |
Polzer, B. et al., 2014 [27][11] | CellSearch | DEPArray | Ampli1 | ERBB2 qPCR (CNV), PIK3CA Sequencing, aCGH | 66 | 510 single CTCs and 189 leukocytes | PIK3CA mutations in exon 9 and 20. Analysis of ERBB2 alterations |
Schneck, H. et al., 2013 [44][8] | CellSearch | NA | Ampli1 | Multiplex PCR, SNaPshot | 44 | NA | PIK3CA mutations in exon 9 and 20, such as E545K and H1047R, were detected, but E542K, E545G and E545A were not found |
Wang, Y. et al., 2018 [22][17] | FACS combined with oHSV1-hTERT-GFP viral infection | FACS | MALBAC | WGS for CTC, WGS and WES for matched primary and metastatic tissue | 8 | 11 single CTCs | SNVs accumulated sporadically among CTCs and matched primary tumors, at least 2 CTCs shared 394 SNVs, SNV mutations in APC and LRP1B genes co-occurred in CTC-shared and bulk tissue, CTC behaviour-related SNVs were verified |
Zou, L. et al., 2020 [21][18] | CellSearch | Micropipetting | MALBAC | WGS (CNV and gene set enrichment analysis) | 2 | Single CTCs, but number is unknown | Different frequencies of CNVs between newly diagnosed and recurrent liver metastasis; similar CNV patterns among isolated CTCs of recurrent BCLM and recurrent liver metastasis; 25 genes were identified as CNV signatures of BCLM, including β-defensins and defensins |
PC or mCRPC | |||||||
Faugeroux, V. et al., 2018 [6][19] | ISET filtration, CellSearch, Rosettesep | Self-seeding microwell chips, FACS, laser microdissection | Ampli1 | WES (10x depth coverage) | 11 | 179 WGA samples or 34 WES | Shared GRM8, TP53 and PTEN mutations in epithelial CTC samples and other CTC-exclusive variants |
Greene, S. B. et al., 2016 [71][20] | Epic Sciences | Eppendorf TransferMan NK4 micromanipulator | SeqPlex Enhanced | Sequencing with Illumina NextSeq500 using a High Output kit in a Paired-End 2x150 format (PE 2x150) (CNV) | 7 | 67 single CTCs | AR amplification and PTEN loss |
Gupta, S. et al., 2016 [72][21] | CellSearch, RBC lysis and CD45 depletion | IE/FACS | RepliGene, WGA4 |
aCGH (CNV) | 16 | 16 CTCs and matched leukocytes | AR amplification in 50% of CTC samples, ERG genomic amplification in 40% of patients, PTEN loss, genomic alteration in chromatin reading and proliferative pathways |
Magbanua, M. J. et al., 2012 [73][22] | CellSearch, IE/FACS | IE/FACS | WGA4 | aCGH | 12 | 9 patient bulk CTCs | Gains in 8q and loss in 8p; gains in the AR region of chr X of CTCs, including AR gains in 78% of cases |
Rangel-Pozzo, A. et al., 2020 [17][23] | ScreenCell filtration | Laser microdissection | Ampli1 | WES | 9 | 21 single CTCs and 4 lymphocytes | Genetic variations in nine telomere maintenance pathways, including telomeric repeat-binding factor 2 (TRF2), SNVs and indels associated with telomere maintenance genes and known cancer drug response; presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway |
Wu, Y. et al., 2016 [4][24] | Density gradient, negative and positive selection with magnetic beads | Laser microdissection | PicoPLEX (<40 cells), WGA2 kit (GenomePlex for microdissected tissues) |
SNP array profiling (CytoSNP-12 and omni1-Quad bead chips, NspI 250k, SNP6.0, and CytoScanHD arrays), Nanostring (nCounter Cancer CN panel) |
8 | 8 disseminated tumor cells (bulk cells) | Gain of Ch 7 and 8q, loss in 8p, 12q23, 10q26, 13q and 16q21. AR gain, TMPRSS2/ERG alterations and MYC and other gained regions, FOXO1 gene deletion |
Lung Cancer | |||||||
He, Y. et al., 2017 [74][25] | CellCollector | CellCollector | REPLI-g | NGS (hotspot panel v2) | 5 | 6 CTCs | 44 cancer-related genes existed in mutations in the analyzed CTCs and some cancer-related mutations were identified in KIT, SMARCB1 and TP53 genes |
Lu, S. et al., 2020 [28][26] | CellSearch | DEPArray | MALBAC, REPLI-g, WGA4, Ampli1 | Targeted sequencing, WES, WGS | 4 | 80 single CTCs and 11 WBCs | Comparative study, MALBAC WGA coupled with LP-WGS is a robust workflow for CNV profiling, but none of the WGA methods achieve sufficient sensitivity and specificity by WES |
Mariscal, J. et al., 2016 [75][27] | CELLection Epithelial Enrich Dynabeads | NA | WTA2 | Gene expression profiling (Agilent 4x44k gene expression arrays), qPCR | 42 NSCLC patients and 16 controls | NA | CTC-specific expression profile associates with the PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1, PTP4A3, LGALS3 and ITGB3 were further validated by RT-qPCR in an independent cohort of NSCLC patients |
Nakamura, I. T. et al., 2021 [13][28] | AutoMACS | DEPArray | SMARTer PicoPLEX | NGS (Todai OncoPanel, AmpliSeq for Illumina comprehensive cancer panel, WGS) and Sanger sequencing |
2 | 40 single floating tumor cells in pleural effusion | EGFR exon 19 deletion was confirmed in 63.2% of samples from case 1, detection of 85% EML4-ALK fusion in case 2, alectinib- resistant mutation of ALK (p.G1202R) in case 2. A BRCA1 truncating mutation and an RAF1 oncogenic mutation were identified |
Ni, X. et al., 2013 [5][29] | CellSearch | Micropipetting | MALBAC | WGS at ∼0.1× sequencing depth and WES for SNV/indel | 11 | 72 single CTCs (including 4 leucocytes) | EGFR mutations (such as one INDEL p.K746_A750del), PIK3CA (such as p.E545K), RB1 (p.R320*) and TP53 mutations (such as p.T155I) were only shared between the liver metastatic tumor and CTCs; gain region in chromosome 8q contains the c-Myc gene; gain in chromosome 5p, which contains the telomerase reverse transcriptase (TERT) gene; chromosomal regions, including 3q29, 17q22, 17q25.3 and 20p13, had significant gain in all 19 CTCs of patients |
Colorectal Cancer | |||||||
Fabbri, F. et al., 2013 [76][30] | OncoQuick | DEPArray | Ampli1 | Sequencing and pyrosequencing | 21 | 16 samples or cases | KRAS gene mutations in 50% of cases. G12C, G12D and G13D-KRAS mutations in one patient in three different groups of CTCs |
Gasch, C. et al., 2013 [19][9] | CellSearch | Micromanipulator TransferMan NK2 | GenomePlex, GenomiPhi | Targeted sequencing for KRAS, BRAF and PIK3CA gene, qPCR for EGFR | 5 | 69 single CTCs | EGFR amplification in 7/26 CTCs, KRAS mutations (G12V) in 33% of CTCs, PIK3CA mutations (E545A and E542K) in 39% of CTCs, no BRAF locus change detected |
Li, R. et al., 2019 [42][31] | Microfluidic chip (SCIGA-chip) | Microfluidic chip (SCIGA-chip) | MDA | Illumina sequencing (SNPs/SVs) | 1 | 2 single CTCs and 1 WBC | A novel method involving all processing steps from blood collection to WGA preparation, 11 shared somatic mutations (e.g., C18orf25, GFM2, DDX60L, etc.) and 153 structure variations were identified |
Pancreatic Cancer | |||||||
Court, C.M et al., 2016 [77][32] | Density gradient and NanoVelcro/LCM microchip |
Laser microdissection | REPLI-g | Sanger sequencing | 12 | 119 single CTCs and 103 WBCs | KRAS mutations in 92% of patients and 33 out of 119 single CTCs sequenced (resulting in a 27.7% detection rate in single CTCs). No KRAS mutants were found in any WBCs |
Melanoma | |||||||
Reid, A. L. et al., 2014 [78][33] | RBC lysis, immune-magnetic beads | NA | REPLI-g | ddPCR and castPCR | 15 | 30 CTCs | Comparative study of ddPCR and castPCR. BRAF-V600E/K mutations were detected |
Ruiz, C. et al., 2016 [79][34] | RBC lysis | Micromanipulator | GenomePlex | CNV analysis | 40 | Single CTCs and WBCs | Deletions of CDKN2A and PTEN; amplifications of BRAF, TERT, MDM2 and KRAS; chromosomal amplifications in chr12, 17 and 19 |
Mixed patient cohort | |||||||
Aljohani, H.M. et al., 2018 [23][35] | RBC lysis, CD45 depletion and EpCam positive selection | FACS | REPLI-g | Sanger sequencing, ddPCR | 10 | NA | Mutations (R34G, E79Q, E82G) in Nrf2 in isolated CTCs, some mutations in the Keap/Nrf2/ARE pathway |
Ferrarini, A. et al., 2018 [80][36] | CellSearch | DEPArray | Ampli1 | WGS (CNAs), aCGH | 3 | 15 single CTCs and 7 WBCs | A large amplification (100 Mbp) on chr 8, including the c-MYC gene, copy number loss was detected in the BRCA2 locus |
Gao, Y. et al., 2017 [81][37] | CellSearch | Micropipetting | MALBAC | WGS and WES for SNV/indels, SVs, CNs | 23 | 97 single CTCs | Homozygous deletion of PTEN; amplification of the MYC gene; 11 focal regions were identified, including well-known tumor suppressor genes or oncogenes, which were deleted or amplified |