Table 1

Detailed technical analysis of different methods used for K-ras detection in sputum

MethodSens.Int. pos. ctrlInt. neg. crtlExt. pos. ctrlExt. neg. ctrlCancerPos. non-cancer/mutation neg. tumourR testRef
*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*nmnmYesYes8/100/5No 53
PCR cloning ASO†nmnmYesYes0/12 54
Enriched-PCR‡npnpYesnm18/375/40yes 55
MASA§nmnmYesYes18/1003/15No 56
PNA-PCR-RFLP¶1/1000npnpYesYes5/510/25 57, 58
Point-EXACCT**1/15 000YesYesYesYes5/110/11Yes 59, 60