The Scale of the Sensitivity Problem:
Finding the Proverbial Needle in a Haystack
Billions of normal cells die every day, eventually liberating their entire DNA into the blood where it is broken down progressively into increasingly minute DNA fragments. The same happens with mutated DNA from cells within small undiagnosed cancers in people who feel perfectly well. The DNA fragments are eventually excreted in the urine.
Typically a ‘Liquid Biopsy’ is an ordinary blood sample that contains within the plasma component an overwhelming concentration of DNA fragments from normal cells, potentially mixed with miniscule amounts of mutated DNA from cancer cells. Urine and PAP smear samples (ThinPrep) can also be used as Liquid Biopsies. The DNA fragments harboring the cancer mutation vary greatly in size and could reside at any point along the DNA fragment. Patent 5
MutantDx’s exquisite sensitivity is due to an ability to detect cancer mutations independent of their location on a fragmented DNA strand, from all sizes of DNA fragments, and at a concentration 1,000 times lower than competing methods — in femtogram quantities.
The clinical benefits of MutantDx’s dramatic technical superiority in sensitivity are threefold:
- Cancer detection at much earlier stages significantly increases the chance of cure in:
- screening for undiagnosed cancer in people who are not yet symptomatic
- screening for recurrence in patients undergoing treatment
- Individuals carrying specific mutations may not be deprived of potentially curative therapy
- No requirement for “enrichment” of the cancer cells, reducing the cost of processing each assay
Detectable DNA fragment size
In blood plasma, circulating cell free DNA (ccfDNA) exists mainly in two fragment sizes: ~ 140 base pairs and 30 base pairs (technically called nucleotides or abbreviated as bp). Technologies currently in use can only detect the larger fragment sizes of ~140 bp. Only MutantDx can detect and confirm mutations in both DNA fragment sizes.
Speed of DNA degradation in the Liquid Biopsy
Breakdown of DNA in the blood is a continuous process, with a rapid breakdown to ~ 140 bp, and then further breakdown to ~ 30 bp, which is the residual state. Present competitor protocols require testing within 4 hours of obtaining the blood sample in order to identify the 140 bp fragments before further degradation makes them undetectable. Since MutantDx has the unique capability of detecting mutations in the residual 30 bp fragments, collection and transportation time to a laboratory is much less critical than with competing methods, providing a significant operational advantage in sample collection logistics.
Location of the mutation
DNA fragments from a cancer can carry the mutation(s) of interest at any location along the length of the fragments. Present methods are unable to detect cancer related mutations harboured toward the ends of the DNA fragments. Only MutantDx can detect mutations anywhere in the DNA fragment, including the extreme ends.
Reference 1 and 3
Limit of detection
The lower limit of detection (LLOD) is determined by four factors:
- the concentration and quantity of DNA fragments carrying the mutation(s)
- the efficiency of extracting DNA from fragments of all sizes including ~30 bp fragments
- the certainty of amplification of all DNA fragments carrying the mutations, and that
- the detection of the mutation(s) can be confidently verified
Only MutantDx has the proprietary extraction, amplification and verifiable detection end point methodologies to accommodate these four important factors.
Detection Index (DI)
A new algorithm has been established to compare the efficiency of molecular technologies for the identification of mutations in cancer tissue (Reference 10 and 11). DI is a measure that combines the relative contributions of the LLOD, amount of DNA, and the ratio of cancer tissue relative to normal tissue in the sample being processed.