Introduction

  • ColoScapeTM Colorectal Cancer Mutation Detection kit is a multiplex real-time PCR based in vitro diagnostic assay for the qualitative and simultaneous detection of colorectal cancer associated mutations in multiple genes that include APC (codons 1309, 1367, 1450 and 876), KRAS (codons 12 and 13), BRAF (codon 600), and CTNNB1 (codons 41 and 45), for Research Use Only
  • The assay is designed to perform on DNA extracted from Plasma or FFPE (Formalin-fixed paraffin embedded). The test identifies the presence or absence of mutations in the targeted region.
  • Genetic mutations detected are:
Genes Exon Amino Acid Change Nucleotide Change Cosmic No.
KRAS 2 G12>A c.35G>C COSM522
G12>R c.34G>C COSM518
G12>D c.35G>A COSM521
G12>C c.34G>T COSM516
G12>S c.34G>A COSM517
G12>V c.35G>T COSM520
G13>D c.38G>A COSM532
G13>C c.37G>T COSM527
G13>R c.37G>C COSM529
APC 15 R876* c.2626C>T COSM18852
E1309fs* c.3921_3925delAAAAG COSM18764
Q1367* c.4099C>T COSM13121
R1450* C.4348C>T COSM13127
CTNNB1 3 p.T41A c.121A>G COSM5664
p.T41I c. 122C>T COSM5676
p.S45P c.133T>C COSM5663
P.S45F c.134C>T COSM5667
P.S45del C133-135delTCT COSM6128
BRAF              15 p.V600E c.1799T>A COSM476
Technology Used:

QClamp® Technology for Mutation Detection

  • Based on xenonucleic acid (XNA) mediated PCR clamping technology
  • When there is a mutation in the target site, and therefore a mismatch, the XNA:DNA duplex is unstable, allowing strand elongation by DNA polymerase
  • Addition of an XNA, whose sequence is a complete match to the wild-type DNA, to a PCR reaction, blocks amplification of wild-type DNA allowing selective amplification of mutant DNA

  

Instructions for use:

  • DNA Isolation
    1. Human genomic DNA must be extracted from fixed paraffin-embedded tissue, tissue, or plasma prior to use. 
    2. Follow the DNA isolation procedure according to manufacturer’s protocol. 
    3. This QClamp assay requires a total of 30 ng of DNA per sample (10ng/reaction). 
    4. After DNA isolation, measure the concentration using fluorometric analysis (i.e., Qubit) and dilute to 5 ng/μl. 
  • Preparation of Reagents
  • Preparation of Assay Mixes
  • Real-Time PCR

Conclusion:

  • All targets can be detected at 1% variant allelic frequency at 5 ng or 10 ng DNA input per PCR reaction.
  • For plasma cfDNA samples, 0.5% variant allelic frequency in all targets except APC 1309 can be detected at 5 ng DNA input.
  • For FFPE DNA samples, 0.5% variant allelic frequency can be detected at 10 ng DNA input.
  • To obtain high sensitivity, the recommended DNA input is a minimum of 10ng/well.

Accuracy:

The analytical accuracy was verified and validated through testing of well-characterized samples with known mutations verified by NGS, Sanger sequencing or ddPCR. Studies were conducted to demonstrate concordance in mutation status of FFPE and plasma samples. The results demonstrated a 100% match between reference methods and the ColoScapeTM kit. 

Precision

Precision of the ColoScapeTM kit was determined with defined analytical levels of genomic DNA with known mutational status and allelic frequencies. Reproducibility is demonstrated based on %CV of Cq values and rate of % correct mutation calls for all assays on two lots and operators for Roche and Bio-Rad instruments.

Summary of reproducibility results

Variation %CV Variation %CV
Intra-assay ≤ 3% Lot to Lot ≤ 4%
Intra-assay ≤ 5% Operator variability ≤ 3%

Analytic Sensitivity and Limit of Detection (LOD)

  • All targets can be detected at 1% variant allelic frequency at 5 ng or 10 ng DNA input per PCR reaction.
  • For FFPE DNA samples, 0.5% variant allelic frequency can be detected at 10 ng DNA input.
  • For plasma cfDNA samples, 0.5% variant allelic frequency in all targets except APC 1309 can be detected at 5 ng DNA input.
  • To obtain high sensitivity, the recommended DNA input is a minimum of 10ng/well.

Analytic Specificity

There were no false positive calls for up to 320ng of gDNA per well and up to 20ng FFPE DNA.

Analytic specificity: cross-reactivity 

Assay Expected Mutations in Tested 50% Templates
APC 1309 APC 1367 APC 1450; KRAS 12 CTNNB1 41 CTNNB1 45; KRAS 13  KRAS 12 KRAS 13 BRAF 600; CTNNB1 45; KRAS 13
APC 1309 + _ _ _ _ _ _ _
APC 1367 _ + _ _ _ _ _ _
APC 1450 _ _ + _ _ _ _ _
CTNNB1 41 _ _ _ + _ _ _ _
CTNNB1 45 _ _ _ _ + _ _ +
KRAS 12  _ _ + _ * + * *
KRAS 13 _ _ _ _ + _ + +
BRAF V600  _ _ _ _ _ _ _ +

Shelf-Life

12 months at production of kit 

Clinical Performance of the Assay

Clinical sensitivity and specificity were tested on the samples extracted from FFPE and plasma of patients with different stages of CRC from normal to advanced adenomas (AA), to colorectal cancer stages 1 through 4. A sample was considered positive if at least one of the target mutations tested positive. The following data was generated during a clinical study of test.

Clinical Sensitivity and Specificity

Test Types of Clinical Samples   Clinical Parameter           
I

 

 

 

   Specificity Sensitivity
CRC (cfDNA) N/A 100%
CRC FFPE N/A 90%
Advanced Adenomas 95% 60%
II FFPE  100% 93%
cfDNA 100% 90%
III cfDNA (Pre-cancer) 92% 70%
Average FFPE  100% 92%
  cfDNA 96% 95%
ENQUIRY NOW