Detection of circulating tumour cells in colorectal cancer: Emerging techniques and clinical implications

Table 1 Techniques for circulating tumour cell isolation, markers, and their limitations

No.	Name	Property	Markers	Limitations	Ref.
1	CellSearch	Isolation by anti-EpCAM antibody coated immunomagnetic beads	EpCAM, CKs, CD45, DAPI	Only suitable for cancer of epithelial origin but not for that undergoing the EMT	[48]
				Cells are not viable after detection
2	AdnaTest	Separation by way of anti-EpCAM and anti-MUC1 antibody coated immunomagnetic beads	EpCAM, MUC1, mucin-1, HER2	Possible false-positive finding due to expression of a selection marker being present in other cells other than CTCs	[49]
				Cells are not viable after detection
3	MACS	Immunomagnetic CTC enrichment by antibodies against cell surface markers	CK19, EpCAM, Her-2, MUC-1 CK7, CK8, CK18, CK19	Lengthy processing time and low sensitivity	[50]
4	MagSweeper (Illumina Inc)	Immunomagnetic isolation of CTC by antibodies against EpCAM and cellsurface markers	EpCAM, CD45, DAPI	Less sensitive during the early stages of tumour development	[51]
		Captured cells are viable with intact RNA
5	CTC Chip	Utilizes bifurcating traps to capture CTCs, release via flow reversal	EpCAM, CKs, CD45, DAPI	Identification of CTCs is lower than other methods	[52]
6	GEM chip	Geometrically enhanced mixing chip structure that allows enhanced capture of CTC on antibody coated surfaces	EpCAM, DAPI, CD45, cytokeratin	Low sensitivity	[53]
7	Onco Quick (Greiner BioOne, Frickenhausen, Germany)	Separation of erythrocytes and some leukocytes from CTC. High sensitivity, Quantification	CCNE2, DKFZp762E1312, EMP2	No morphology confirmation; not really capture CTCs	[54]
8	ISET (Rarecells Diagnostics)	Rapid processing; non-antigen dependent; Filter based approach	CKs, EGFR, VE-cadherin, Ki67	Size-dependent, manual processing	[55]
9	EPISPOT	Removes leukocytes via CD45 depletion	CD45, CK19, mucin-1, cathepsin-D	Problem arises when antigen levels are lower or binding efficiency is reduced	[56]
		Can detect viable CTCs
10	Ficoll + RT-PCR	Separation of CTC based on size dependent enrichment. High Sensitivity	CK-19, HER2, h-MAM, CEA, maspin, GABA A, B726P	No morphology confirmation	[57]
11	Cyttel Method	Negative immune-magnetic selection of WBC (CD45 antibody)-High detection rate	CD45	-	[58]
12	MetaCell	Size-based enrichment and separation for viable CTCs	CK-18, -19, -20, CK-7, EPCAM, MUC1, HER2, EGFR	Lengthy processing time	[59]

MACS: Magnetic-activated Cell Sorting; CTC: Circulating tumour cell; RT-PCR: Real-time polymerase chain reaction.

Table 2 Studies showing postoperative isolation of circulating tumour cells in colorectal cancer–markers, techniques, and clinical implications

No.	Technology	Markers	Number of patients	TNM stage	Correlation	Clinical significance	Ref.
1	CellSearch system	EpCAM	164	I-III	With stage	N/A	[60]
		EpCAM	24	IV	With therapy response	May be used in monitoring response to therapy	[61]
		EpCAM	97	II	With stage	Correlates with stage	[62]
		CD133+, CD54+, CD44+	15 (nmCRC); 95 (mCRC)	I-IV	≥ 5 CTCs were 8 times more likely to develop distant metastasis. CTC counts show good correlation with colorectal neoplasm	Independent prognostic marker for nmCRC	[63]
		hTERT, CK19, CK20, CEA	438	I-III	-	Poor relapse free survival	[64]
		hTERT, CK19, CK20, CEA	157	I-III	With stage	Poor relapse free survival and overall survival	[65]
		Survivin, CK20 and CEA	156	I-III	With stages (Duke’s) and lymph node metastasis.	Useful as an adjunct in detection of CRC patients	[66]
		CD133, CEA, CK20, CK19,	197	II-III	CEA/CK/CD133 expression and stage (Duke’s)	Prognostic significance (Duke's stages B and C)	[44]
		hTERT, CK-19, CK-20, CEA, GAPDH and mRNA	72	I-IV	CEA, mRNA: With stage, vascular invasion, and postoperative metastasis	Prognostic and predictive	[67]
2	Flow-cytometry with immunofluorescence	CTCs	18	I-III	With stage and also detected in an early cancer stage.	Predictive	[68]
3	Pyrosequencing	KRAS (Codon 12/13)	26	IV	No association	Prognostic	[69]
4	MetaCell separation method	CTCs	98	I-IV	CTC-positive in 83%	Prognosis and predictive	[70]
					CTC-negative in 17%
5	Mag Sweeper	PIK3CA	242	-	Mutational discordance found between CTCs, DTCs, and metastases, and among CTCs; DTCs from this patient propagated in vitro contained a PIK3CA mutation	Investigating new drug therapies	[71]
6	CTC-Chip	EpCAM, HER2, and EGFR	-	-	Efficiency of 87.5%	In situ protein expression, and culture CTCs from the same set of cells	[72]

CRC: Colorectal cancer; DTC: Disseminated tumour cells; GAPDH: Glyceraldehydes 3-phosphate dehydrogenase; nmCRC: Non-metastatic CRC; mCRC: Metastatic CRC.

Table 3 Circulating tumour cells in metastatic vs non-metastatic colorectal cancer

No.	Type of CRC	Markers used	Detection method used	Relevance	Clinical implications	Limitations of the study	Ref.
1	nmCRC	CEA, CA19-9, CA72-4	Cyttel	Diagnostic/prognostic/predictive	Combination of CTCs and CEA: Diagnostic and prognostic indicators	Small sample size, weak power of the study	[73]
2	mCRC	CK, CD45	Immunomagnetic separation	Prognostic/predictive	The number of CTCs before and during treatment is an independent predictor of PFS and OS in patients with mCRC	The baseline unfavourable CTC was low (26%) and overall CTC yield was less than in other epithelial cells	[74]
3	mCRC	ALDH1, CD44, CD133, MRP5, Survivin	qRT-PCR	Prognostic	Poor prognosis and chemo therapy non-responsiveness	Require further molecular analyses of CTCs for selection of targeted agents	[75]
					Survivin and MRP5 selection of mCRC patients resistant to 5-FU and L-OHP
4	mCRC	CEA	Cyttel method, immunofluorescence in situ hybridization technologies (imFISH)	Prognostic	PFS, OS	Small sample size	[6]
						Lack of dynamic enumeration of CTCs
5	mCRC	VEGF, CD133+, CD34+/KDR + EPC, CD-34-VEGFR2	Flow cytometry/IHC	Prognostic	Treatment response; PFS, OS	-	[76]
6	nmCRC	CD133, CD166, CD44, EpCAM, ALDH1	Tissue microarray, IHC	Prognostic	No association with poor clinical response; OS	Treatment information was missing (local recurrence, distant metastasis, and postoperative therapy)	[77]
7	nmCRC	CK19, MUC1, CD44, CD133, ALDH1	Flow-cytometry, CellSearch, Cytomorphology, qPCR	Prognostic	May be useful as a therapeutic target; PFS, OS	-	[78]

CRC: Colorectal cancer; nmCRC: Non-metastatic CRC; mCRC: Metastatic CRC; OS: Overall survival; PFS: Progression-free survival; IHC: Immunohistochemistry.