Our SOlution

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Blocking PCR is the concept of selectively replicating cancer DNA by preventing the replication of normal DNA. The purpose is to increase the amount of cancer DNA relative to the amount of normal DNA. For example, a patient's blood may only contain 0.005% cancer DNA, with the rest being normal DNA. After all of the DNA is removed from the blood, Blocking PCR selectively replicates the cancer DNA. This increases the frequency of cancer DNA from >0.005% to >99%.

Blocking PCR solves the two major issues with liquid biopsy: 

1. Large amount of DNA sequencing needed to find low frequency cancer DNA
2. High error rate of sequencing by synthesis. 

By increasing the frequency of cancer from 0.005% to >99% the amount of DNA required for sequencing is significantly reduced. Methods that do not use Blocking PCR have to sequence >100,000 copies of DNA, where Blocking PCR would only require 100 copies to sequenced.

By increasing the frequency of cancer from 0.005% to >99% the error rate of sequencing by synthesis is no longer relevant. The error rate of sequencing by synthesis is >0.1% with a standard deviation of >0.1%. So any sample with >5% cancer DNA, is too many standard deviation away for a false positive to be caused by sequencing error. The main source of error, at the detection step, is effectively removed.

The benefits of Blocking PCR have long been known. The first Blocking PCR paper was published in 1996. Since then, there have been numerous blocking methods such as PNA Clamping, LNA Blocking, ICE COLD PCR (Precipio), XNA Clamping (Diacarta), Switch Blocking (Aegea) and Blocker Displacement Amplification (NuProbe). None of these methods have been widely adopted despite the clear advantages of Blocking PCR and the growing need for accurate low frequency DNA mutation detection.

The methods that currently dominate the market for low frequency cancer detection are unique dual indexes (Natera) and digital PCR (Bio-Rad). Blocking PCR is something that could replace these existing technologies or be added to these existing approaches. Bio-Rad recently licensed NuProbe's blocking method and has launched a cancer diagnostic test, which combines digital PCR with blocking PCR.

SentryDx took a unique approach to developing a new blocking method, compared to the other blocking methods previously listed. NuProbe, Diacarta and the others, all advertise the high sensitivity of their respective system (e.g. the ability to detect a single copy of cancer DNA or a limit of detection of 0.005%). However, a lack of sensitivity is not why these blocking methods failed to commercialize successfully. The first blocking method in 1996 could detect a single copy of cancer DNA. The problem is that when the method is too good at blocking, normal DNA is never replicated enough to detect. This results is a lack of performance and quality control. SentryDx has a unique way to overcome this issue and a patent has already been issued.

The issue of blocking too well, can most easily be demonstrated by the Bio-Rad and NuProbe partnership, which launched their first cancer diagnostic assay for ESR1 mutations. Figure 1 of Bio-Rad document 10000171375 shows how the test has to be split into two separate components. The User Manual states, "the ddPLEX Mutation Detection Assay (#12019648) comprises two different assays. The wild-type assay measures the wild-type loci in three exons of the ESR1 gene (see Table 4), The mutant assay quantifies seven common variants (E380Q, S463P, L536R, Y537C, Y537N, Y537S, and D538G)". This splitting approach highlights that the NuProbe blocking method cannot detect cancer DNA and normal DNA (wild-type) at the same time. This split approach is not found in other Bio-Rad cancer tests, which do not use NuProbe technology. The inability to detect both cancer DNA and normal DNA at the same time, is why Blocking PCR has never been widely adopted. The solution developed by SentryDx is why our technology is special and why SentryDx has the potential to greatly improve cancer diagnostics.

SentryDx presented a poster at AMP 2024, which demonstrates the amazing results that this blocking method can achieve. The Results section demonstrates how the method can accurately detect a sample which is 0.01% cancer DNA or 100% normal DNA, using the same process. This is the key differentiator for the SentryDx blocking method.

The SentryDx method has also been independently verified by another laboratory. The other laboratory was able to follow the provided protocols and achieve the same performance as demonstrated in the AMP poster. Independent verification is a major step toward commercial success.