Detailed technical analysis of different methods used for K-ras detection in sputum
| Method | Sens. | Int. pos. ctrl | Int. neg. crtl | Ext. pos. ctrl | Ext. neg. ctrl | Cancer | Pos. non-cancer/mutation neg. tumour | R test | Ref |
|---|---|---|---|---|---|---|---|---|---|
| *ASO, allele specific oligonucleotide hybridisation; case selection: only K-ras positive adenocarcinomas; threshold for number of spots to be called positive not mentioned. †Sputum samples with PAP III–V, no direct relation to K-ras positive cancer known. ‡Twenty cycles of PCR, then digestion with BstN1 for a cut in wild-type alleles and subsequently another “x” number of cycles; all squamous cell carcinomas. Repeated samples performed three times, no further details, one first analysis requires 2 μg DNA. §MASA, mutation allelic specific amplification; PCR 1 × 32 cycles; Positive cancer cases include 11/59 adenocarcinomas and 5/32 squamous cell carcinoma (SQCC) cases; 5 cases in sputum without K-ras positivity of primary tumour; Int. neg. crtl nm implies not possible because a mixture of mutant primers was used. ¶From primary tumour K-ras status not known; 4/22 SQCC, 1 adenocarcinoma, 2 large cell carcinoma cases. Quote58: use of 2 PCR steps with PNA-PCR-RFLP instead of 3 for PCR-RFLP reduces Taq polymerase borne errors; 54 cycles; concordant with washing sample of same patient. **Paraffin wax embedded sputum from patients with adenocarcinoma: 5/11 cases with K-ras positive and 0/11 cases with K-ras negative tumours.61 | |||||||||
| Sens., minimum target frequency (also called sensitivity of the technique); Int. pos. ctrl, internal positive control; Int. neg. crtl, internal negative control; Ext. pos. ctrl, external positive control; Ext. neg. ctrl, external negative control; Pos. cancer, number and percentage of positive lung cancer cases; Pos. non-cancer, number and percentage of positive cases in patients without lung cancer; R test, test repeated?; nm, not mentioned; np, not possible; –, not done. | |||||||||
| PCR cloning ASO* | nm | nm | Yes | Yes | 8/10 | 0/5 | No | 53 | |
| PCR cloning ASO† | nm | nm | Yes | Yes | 0/12 | – | – | 54 | |
| Enriched-PCR‡ | np | np | Yes | nm | 18/37 | 5/40 | yes | 55 | |
| MASA§ | nm | nm | Yes | Yes | 18/100 | 3/15 | No | 56 | |
| PNA-PCR-RFLP¶ | 1/1000 | np | np | Yes | Yes | 5/51 | 0/25 | 57, 58 | |
| Point-EXACCT** | 1/15 000 | Yes | Yes | Yes | Yes | 5/11 | 0/11 | Yes | 59, 60 |