Table 1. Genomic heterogeneity in CTCs.
| #CTC | #Pts | Isolation | Analysis | Targets | Heterogeneity | Ref. |
|---|---|---|---|---|---|---|
| Lung cancer | ||||||
| n.s. | 32 | MF (ISET) | FA-FISH | ALK rearrangements | 18 ALK+ patients exhibited between 7 and 24 CTCs/ml, mean percentage of ALK-rearranged CTCs was 63% (range 28-100%). All ALK– patients had < 4 rearranged CTCs. | 89 |
| n.s. | 5 | MF (ISET) | FA-FISH | ALK rearrangements | 5 patients showed ALK-gene rearrangements in all CTCs (100%), while in the primary tumor only half of the tumor cells show these rearrangements. | 91 |
| 177 | 1 | microfluidics + cytospin | FISH | ALK rearrangements | 25% of the total 177 CTC of 1 patient harbored ALK-gene rearrangements, and 54% of the 200 primary tumor cells did. | 90 |
| n.s. | 8 | MF (ISET) | FA-FISH | ROS1 rearrangements | ROS1 rearrangements were detected in the CTCs of all 4 ROS+ patients. ROS1 copy number was heterogeneous within these CTCs and increased at time of disease progression. | 95 |
| 8 | 1 | CS + MM | WES | CNA; mutations; indels | CNA show inter-CTC homogeneity, and represent the metastatic tumor. SCLC and NSCLC can be differentiated based on CNA-profile. Mutations and indels were highly heterogeneous in all CTCs. | 35 |
| 8 + pools | 2 | CS + DEPArray | WGS; TAS | CNA; TP53, RB1 mutations | CNA strongly correlated, but 1 of 6 CTC harbored substantial CNA differences. TP53 and RB1 mutations were homogeneous. | 112 |
| 1 pool | 4 | microfluidics | Allele-specific PCR | EGFR mutations and CNA | Temporal heterogeneity in EGFR mutations. Genotypes of enriched CTC fractions evolved during therapy, with consistent presence of the primary EGFR activating mutation and the emergence of a drug-resistant mutation. | 65 |
| Colorectal cancer | ||||||
| 37 | 6 | CS + MM | aCGH; Panel | CNA; 68 CRC-related gene panel | Multiple CRC related CNA and mutations were found in CTC and tissue samples. Various CTC-specific mutations were detected, but retraced at subclonal level by ultra-deep sequencing of the tissue samples. Inter-CTC heterogeneity, with some private mutations. | 33 |
| 741 | 33 | CS + MM | qPCR; TAS | EGFR CNA; PIK3CA, KRAS, and BRAF mutations | CN-gain of EGFR was found in 27% of CTCs of 3 patients, 1 patient had KRAS mutations in 33% of CTCs, 39% of CTCs of 4 patients harbored PIK3CA mutations. | 105 |
| 126 | 31 | CS + MM | TAS | TP53, KRAS and BRAF mutations | CTCs were analyzed of 18 patients. 6 patients harbored heterogeneous CTC populations. | 107 |
| pools | 21 | DGC + DEPArray | TAS; PyroSeq | KRAS mutations | In 1 patient, 3 pools of CTCs had different mutational statuses, two mutations were found in the first pool and another mutation in a second pool of isolated CTCs. | 108 |
| pools | 2 | CS enriched | qPCR | KRAS mutations | Temporal heterogeneity: enriched CTC fractions exhibited different mutational status of KRAS during treatment. | 109 |
| Prostate cancer | ||||||
| n.s. | 49 | CS | On-chip FISH | ERG rearrangements;PTEN and AR CNA | FISH on CTCs revealed homogenous ERG rearrangements but heterogeneous AR amplifications and PTEN deletions. | 96 |
| n.s. | 77 | CS + cytospin | FISH | AR and MYC CNA | There was considerable variability in the morphology of CTCs in individual patients. 1 patient showed heterogeneity of FISH patterns, with AR amplification in a subset of CTCs, but all with high copy number gain for MYC. | 117 |
| n.s. | 7 | DGC + cytospin | FISH | BRCA1 CNA | In 4 of 7 patients, BRCA1 losses appeared in a fraction of CTCs. | 118 |
| pools | 9 | IE/FACS | aCGH | CNA | CTCs from all patients revealed a wide range of CNA. Replicate CTC isolates where comparable showing gains in the CCND1 and AR locus. | 114 |
| 41 | 1 | HD-CTC + MM | WGS | CNA | Three different clonal lineages were found. Clone B was present subclonally at first blood draw, but demonstrated outgrowth in the third blood draw. A third clone emerged at fourth blood draw. | 80 |
| 19 + 10 | 2 | MagSweeper + MM | WES | Somatic SNV | Although non-uniform coverage, a heterogeneous mutation profile was detected in single CTCs. When pooling the CTC data, found SNVs were comparable to the primary tumor. | 34 |
| Breast cancer | ||||||
| 261 + pools | 42 | CS + DEPArray | aCGH; qPCR; TAS | CNA; ERBB2 CNA; PIK3CA mutations | 2 patients had heterogeneous PIK3CA mutational status in their single and pooled CTCs. 10 of 16 patients harboring PIK3CA mutations showed molecular heterogeneity based on CNA. ERBB2 amplification was uniformly detected in all CTCs of 7 patients. | 101 |
| 26 | 12 | CS + flow sorting (MoFlo XDP) | aCGH; qPCR; TAS | CNA; CCND1 CNA; PIK3CA mutations | CNA were found breast cancer related in all CTCs, but differences in CNA between related CTCs were present in all cases. 1 patient harbored a mutation in exon 20 of the PIK3CA gene in both CTCs and 1 patient harbored another PIK3CA mutation in 1 of 1 CTCs. | 100 |
| 147 + pools | 26 | CS + DEPArray | TAS | PIK3CA hotspots | 11 of 26 patients were found to harbor a heterogeneous PIK3CA mutational status in their CTC compartment. | 102 |
| 115 + pools | 18 | CS + DEPArray | TAS | PIK3CA hotspots | 3 patients were homogeneously mutated in all CTCs. 1 patient was found to have three different PIK3CA mutations. | 104 |
| 185 | 17 | MagSweeper + MM | TAS | PIK3CA hotspots | 1 patient harbored a heterogeneous CTC compartment based on PIK3CA status. | 103 |
| 11 + pools | 2 | CS + DEPArray | TAS | TP53 mutations | In one patient, 2 of 6 single CTC harbored two different TP53 mutations. In the second patient, 3 of 5 single and 5 of 6 clusters of CTCs showed a TP53R110delC mutation. | 111 |
| 402 | 3 | DGC + cytospin | IF/FISH (BioView) | EGFR CNA | 10 of 91 ALDH1+/HPSE+ cells showed EGFR amplification. This was 19 of 311 in the ALDH1–/HPSE+ population. | 50 |
| 31 + pools | 1 | CS ór DGC + MM | WGS; aCGH | CNA | CNA show homogeneity within all isolated CTCs. | 36 |
| n.s. | 3 | IE/FACS | aCGH | CNA | Temporal heterogeneity: Serial testing of enriched CTC populations revealed numerous additional CNA beyond the baseline profile. | 116 |
| Melanoma | ||||||
| 24 + 18 | 2 | Microfluidic + LCM | TAS | BRAF mutations | Consistency in the BRAFV600E mutation, and in accordance with the primary tumor. | 110 |
| 15 | 7 | IM + MM | CGH | CNA | In 5 of 6 patients with ≥ 1 isolated CTC, hierarchical clustering showed a clonal origin. | 115 |
| Multiple cancers | ||||||
| n.s. | 20 | IM + cytospin | FISH | CNA | 6 patients had a homogeneous pattern of aneusomy in all CTCs. In 10 patients a heterogeneous pattern was observed, including 6 cases with two distinct clones. | 49 |
Abbreviations: aCGH, array comparative genomic hybridization; CNA, copy number alterations; CS, CellSearch enrichment; CTC, circulating tumor cell; DGC, density gradient centrifugation; FA-FISH, filter adapted fluorescent in situ hybridization; HD-CTC, high-definition CTC assay; IE/FACS, immunomagnetic enrichment and fluorescence-activated cell sorting; IF, immunofluorescence; IM, immunomagnetic enrichment; LCM, laser capture microscopy; MF, microfiltration; MM, micromanipulation; TAS, targeted amplicon sequencing; qPCR, quantitative polyclonal chain reaction; WES, whole exome sequencing; WGS, whole genome sequencing; n.s., not specified.