Table 2
The detection of breast cancer cells in blood using multiple molecular markers
| Markers | Amplification method | PCR cycling conditions | Sensitivity | Specificity in control blood | Combined positivity | Ref |
|---|---|---|---|---|---|---|
| βhCG, β chain of human chorionic gonadotrophin; β-NAcGAT, β-N-acetylgalactosaminyl transferase; CEA, carcinoembryonic antigen; CK, cytokeratin; c-met, hepatocyte growth factor receptor; EGFR, epidermal growth factor receptor; MAGE.A3, melanoma associated antigen; m’globin, mammaglobin; MNC, mononuclear cells; MUC1, membrane associated mucin 1; ND, not determined; PBL, peripheral blood leucocytes; PBMN, peripheral blood mononuclear cells; PIP, prolactin inducible protein; PSE, prostate specific ets factor; Q, quantitative; RT-PCR, reverse transcription polymerase chain reaction. | ||||||
| CEA, CK19, maspin | Semi-nested or nested RT-PCR | CEA: one 30 and one 20 cycle reaction | CEA/maspin: 10 T47-D cells/106 MNC | CEA: specific | 50% of samples were positive for CK19 and CEA | 28 |
| CK19: two 35 cycle reactions | CK19: 1 T47-D cell/106 MNC | CK19 and maspin: not specific | 83% were positive for at least one of the two markers CEA and CK19 | |||
| Maspin: two 35 cycle reactions | 90% and 70% of maspin positive samples were positive for CEA and CK19 respectively | |||||
| M’globin, CK19 | Nested RT-PCR | One 43 and one 40 cycle reaction | 1 tumour cell/ml of blood | Specific | 1 sample positive for both markers | 49 |
| β-hCG, c-Met, MAGE-A3, β-NAcGAT | RT-PCR | One 35 cycle reaction for each marker | 1–5 tumour cells in 107 PBL for each marker | Specific | 49% patients positive for 1 marker | 73 |
| Each marker could be detected at total RNA concentrations ⩾1.0 ng/ml | 15% positive for 2 markers | |||||
| 3% positive for 3 markers | ||||||
| 2% positive for all four markers | ||||||
| CEA, CK19, CK20, GA733.2, MUC1 | RT-PCR and southern blot | One 35 cycle reaction for each marker | 10 ng RNA of each of the markers could be detected by southern blotting | CK20: specific | ND | 74 |
| All other markers: not specific | ||||||
| MUC1, CK19, CD44 | RT-PCR and nested RT-PCR | MUC1: one 30 cycle reaction | MUC1: 1 MCF-7 cell/ml of blood | CK19: specific | ND | 75 |
| CK19: one 40 cycle reaction and one 35 cycle reaction | CK19: 104 MCF-7 cells/10 ml of blood | MUC1 and CD44: not specific | ||||
| CD44: one 20 cycle reaction and one 30 cycle reaction | ||||||
| Maspin, CK19 | Nested RT-PCR | Two 35 cycle reactions | 10 MCF-7 cell in 106 PBMN | Maspin: specific | ND | 76 |
| CK19: not specific | ||||||
| CK19, MUC1, CEA | RT-PCR, semi-nested, and nested RT-PCR | CK19: One 35 and one 30 cycle reaction | MUC1 and CK19: 1 T47-D cell/106 PBMN | CK19 and CEA: specific | All CEA positive patients were positive for CK19. | 77 |
| MUC1: one 35 cycle reaction | CEA: 1 T47-D cell/5×105 PBMN | MUC 1: not specific | Not all CK19 positive patients were CEA positive | |||
| CEA: one 20 cycle reaction and one 30 cycle reaction | ||||||
| CK19, MUC1, CEA | RT-PCR, semi-nested, or nested RT-PCR | CK19: two 35 cycle reactions | CEA (A): 1 MCF-7 cell in 102 PBMN; CEA (B): 1 MCF-7 cell in 103 PBMN | CK19 and MUC1, not specific | ND | 78 |
| MUC1: one 35 cycle reaction | CEA: specific unless treated with G-CSF | |||||
| CEA (A): one 20 cycle reaction and one 30 cycle reaction | ||||||
| CEA (B): 33–40 cycles | ||||||
| M’globin, CK19, EGFR | Nested RT-PCR | M’globin and EGFR: two 40 cycle reactions | 1 MD-MBA-361 cell in every 106 PBMN | M’globin: specific | 5 patients m’globin/CK19 positive and 2 patients positive for all 3 markers | 79 |
| CK19: two 30 cycle reactions | CK19 and EGFR: not specific | |||||
| Maspin, m’globin, CK19, CK20, CEA, MUC1, EGFR | Nested RT-PCR | MUC1/EGFR/CEA: two 30 cycle reactions | 1–10 MCF7 or SKBR-3 cells/106 PBMN cells for all assays | Maspin, CK20, and m’globin: specific | ND | 80 |
| CK19: two 35 cycle reactions | CK19, CEA | |||||
| CK20: two 32 cycle reactions | MUC1, EGFR: not specific | |||||
| Mammaglobin: one 32 cycle and one 35 cycle reaction | ||||||
| Maspin: one 30 cycle and one 35 cycle reaction | ||||||
| Maspin, m’globin, c-ErbB2 | Q RT-PCR | One 40 cycle reaction for each marker | Maspin and m’globin: 1 MCF-7 cell in 103 PBMN | Maspin and m’globin: specific | ND | 81 |
| Maspin: 1 ng MCF-7 total cDNA | c-ErbB-2: not specific | |||||
| M’globin: 100 pg MCF-7 total cDNA | ||||||
| CK19, CK20, EGFR | RT-PCR | CK19: one 40 cycle reaction | ND | CK19 and CK20: not specific | 5% positive for both CK19 and EGFR | 82 |
| CK20: one 42 cycle reaction | EGFR: specific | |||||
| EGFR: one 33 cycle reaction | ||||||
| CK19, CK20, β-hCG | RT-PCR | Not detailed | CK19/CK20: 1 MCF-7 cell in 105 HL-60 cells; β-hCG 1: MCF-7 cell in 106 HL-60 cells | CK19: not specific | CK19/CK20: 9.72% | 83 |
| CK20 and β-hCG: specific | CK20/β-hCG: 13.9% | |||||
| CK19/β-hCG: 18.1% | ||||||
| MUC1, CK19, PIP, m’globin, m’globin B, PSE | Q RT-PCR | One 40 cycle reaction for each marker | 1 MDA-361 cell in 5×108 PBL | MUC1: not specific | 20% patients expressed 1 marker | 84 |
| CK19, PIP, m’globin | 25% patients expressed 3 markers | |||||
| m’globin B, PSE: specific | 20% patients expressed 4 or more markers | |||||
