Table 1.
KRAS mutations in 300 consecutive samples analyzed by Sanger sequencing and ASLNAqPCR.
Table 2.
BRAF mutations analysis in 201 consecutive samples analyzed by Sanger sequencing and ASLNAqPCR.
Table 3.
Primers and beacon probes.
Figure 1.
Diagram illustrating ASLNAqPCR.
Left side: a single mismatch of the LNA modified primer does not allow PCR amplification. Right side: in case of a perfect match, the Taq polymerase extends the DNA strand and the amplicon is detected by the internal LNA modified beacon probe.
Table 4.
PCR conditions for Sanger sequencing and ASLNAqPCR.
Figure 2.
Standard curve titration of ASLNAqPCR for KRAS.
Serial dilution of the KRAS G12V mutated SW620 cell line DNA in wild type DNA. Gray squares correspond to 50%, 20%, 10%, 5%, 1%, 0,1%, 0.01% mutant to wild type DNA ratios (duplicate samples). The titration slope is −3.076, R2 is 0.991 (top right), corresponding to a PCR efficiency of 111.3%.
Figure 3.
Standard curve titration of ASLNAqPCR for BRAF.
Serial dilution of the BRAF V600E mutated OCUT cell line DNA in wild type DNA. Gray squares correspond to 50%, 20%, 10%, 5%, 1%, 0,1%, 0.01% mutant to wild type DNA ratios (duplicate samples). The titration slope is −2.985, R2 is 0.991 (top right), corresponding to a PCR efficiency of 116.2%.
Table 5.
Frequence of specific KRAS and BRAF mutations cases analyzed by SSEQ and ASLNAqPCR.
Table 6.
KRAS mutations in primary colon carcinoma (n = 163) compared with data reported in the literature
Table 7.
KRAS Pyrosequencing analysis of cases with discordant results between ASLNAqPCR and Sanger sequencing.
Table 8.
BRAF Pyrosequencing analysis of cases with discordant results between ASLNAqPCR and Sanger sequencing.
Figure 4.
ASLNAqPCR and corresponding Sanger sequencing of four representative tumor samples analyzed for KRAS mutations.
Sample A is wild type, samples B, C and D are KRAS G12D mutated with varying amounts of tumor vs. non neoplastic cells; assuming that KRAS G12D is heterozygous, quantitation of mutated DNA by ASLNAqPCR (ΔCT method) is consistent with 70% of mutated cells in sample B, 40% of mutated cells in sample C, 4% of mutated cells in sample D; in sample D the KRAS G12D mutation is detected only by the ASLNAQPCR due to its high analytical sensitivity.
Figure 5.
ASLNAqPCR and corresponding Sanger sequencing of four representative tumor samples analyzed for the BRAF V600E mutation.
Sample A is wild type, samples B, C and D are BRAF V600E mutated with varying amounts of tumor vs. non neoplastic cells; assuming that BRAF V600E is heterozygous, quantitation of mutated DNA by ASLNAqPCR (ΔCT method) is consistent with 75% of mutated cells in sample B, 30% of mutated cells in sample C, 3% of mutated cells in sample D; in sample D the BRAF V600E mutation is detected only by the ASLNAQPCR due to its high analytical sensitivity.
Figure 6.
KRAS mutations identified by ASLNAqPCR but not by Sanger sequencing. A
, Hematoxylin and Eosin (H&E) stained section (X100) of the area of case 13 of Table 7 (rectal adenocarcinoma treated with preoperative chemo– and radiation therapy) dissected for DNA extraction with a tumor vs. non neoplastic cell ratio of ∼10%, below the analytical sensitivity threshold of Sanger sequencing. B, ASLNAqPCR of case 13 of Table 7 shows a KRAS G12V mutation with a mutated/wild type ratio of 4%, corresponding to 8% mutated cells, assuming that the mutation is heterozygous; this is consistent with the mutation being present in the large majority of neoplastic cells. C, H&E stained section (X100) of the area of the colonic adenocarcinoma case 1 of Table 7, dissected for DNA extraction with a tumor vs. non neoplastic cell ratio of ∼70%. D, ASLNAqPCR of case 1 of Table 7 shows a KRAS G12D mutation with a mutated/wild type ratio of 1.5%, corresponding to 3% mutated cells, assuming that the mutation is heterozygous; this is consistent with a small KRAS G12D mutated subclone corresponding to ∼4% of the neoplastic cells.
Table 9.
Statistical measures of performance for Sanger sequencing and ASLNAqPCR.