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German Team Combines Wild-Type Blocking PCR, HRM to Detect Low-Level Colon Cancer Mutations in Stool

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A team from the University of Potsdam in Germany has combined wild-type blocking PCR using locked nucleic acids with high-resolution melting analysis to detect DNA mutations of unknown type and position in a background of up to 10,000-fold wild-type DNA.

The researchers also used the method, dubbed WTB-HRM, to enrich and detect such known and unknown mutations in the cancer-related adenomatous polyposis coli, or APC, gene in tissue and stool samples of colorectal cancer patients.

As such, the group believes it has laid the groundwork to develop a non-invasive diagnostic test that could be used to screen for early colorectal cancer as well as precursor lesions by detecting cancer-initiating mutations in APC and other genes similarly associated with colorectal cancer, such as KRAS and BRAF.

The scientists, primarily from the Department of Nutritional Toxicology at the University of Potsdam, described their method in a paper published last week in Cancer Prevention Research.

Tumor cells are released into stool from the surface of precancers and early-stage colon cancers, but detecting these mutations in stool samples containing a huge amount of wild-type DNA is challenging, the researchers explained.

Precancer cells containing these genetic variations can also be detected in blood – which is the basis of several commercial colon cancer screening tests currently under development – but researchers believe that detecting the mutations in such cells from stool samples could enable successful screening in the very earliest stages of disease.

In addition, most early-detection tests have focused on KRAS and BRAF mutation analysis. However, mutations in the APC gene have also been implicated in the development of colon cancers, but have been notoriously difficult to detect due to the fact that different mutation types can occur simultaneously in one of several mutational "hotspots" within the gene.

"For this reason, detection methods using mutant-specific probes are inappropriate in this case, and although APC is the most frequently mutated gene in colorectal cancer, there is no sensitive commercial test for APC mutation analysis available" – a need that the researchers sought to address with their assay technique, they wrote in their paper.

"Our aim was to develop a technique for the ultra-sensitive detection of known as well as unknown gene mutations that occur during the formation and progression of colon cancer," Bettina Scholtka, an assistant professor of nutritional toxicology at the University of Potsdam and the corresponding author on the paper, told PCR Insider in an email.

"For this purpose we tried various combinations of methods for mutation enrichment and mutation detection," Scholtka added. "The difficulty is to combine methods that are on the one hand sensitive enough for non-invasive screening and able to detect unknown or unexpected mutations, and on the other hand are automatable as well."

To tackle this problem, the group developed a two-step assay method that uses locked nucleic acid, or LNA, blocker molecules to suppress wild-type DNA followed by further enrichment and detection of mutations using high-resolution melting analysis.

More specifically, Scholtka and colleagues analyzed the mutation cluster regions of the APC gene by subdividing it into four overlapping segments of 200 to 300 base pairs each that were analyzed separately using oligonucleotide primers that the group had previously developed.

They designed and calculated melting conditions of LNA blocker molecules using the LNA Oligo design tool, available online from Exiqon. LNA blockers hybridized exclusively with wild-type DNA in the space of the respective mutational hotspots in corresponding gene segments, and one LNA blocker was included in every reaction to suppress wild-type DNA amplification.

Further, the researchers carried out high-resolution melting analysis on a Roche LightCycler 480 II instrument running LC 480 Gene Scanning Software to further enrich and detect mutations in specimens previously amplified by the PCR assay.

"The use of LNA blocker molecules in the wild-type blocking PCR step offers the possibility to enrich the sample not only for different types of expected mutations but also for unknown mutations being situated in the same PCR product by suppressing wild-type amplification," Scholtka explained.

"By combining WTB-PCR with HRM we could achieve the desired detection limit of a 0.01 percent mutation level in excess wild-type DNA," she added. "This is the necessary detection limit for the purpose of non-invasive stool screening when [taking] into account that the ratio of mutated human DNA to total DNA [in stool] is assumed to be in the range of 1 in 10,000 to 1 in 100,000."

The researchers first tested their technique on 80 human colon tissue samples representing cancers and precancers, and were able to detect APC mutations – including previously unknown variations – in 41 of the samples. In contrast, direct sequencing was only able to detect mutations in 28 samples.

Scholtka and colleagues then analyzed 22 stool samples from patients whose colon tissues had APC variations, and nine control stool samples from patients whose colon tissues did not have APC variations. Here, they were able to detect APC mutations in all but one of the 22 stool samples in which the corresponding tissue sample had variations.

Finally, the team used its method to detect variations in the KRAS gene using 20 human colon tissue samples to demonstrate that the WTB-HRM technique can be used to detect variations in genes other than APC.

"Because of its exceptionally low detection limit, the WTB-HRM method could facilitate noninvasive screening for early colorectal cancer as well as for precursor lesions by detecting cancer-initiating gene mutations in body fluids like stool," the researchers wrote. As such, they noted, it would also be interesting to investigate "whether those mutations indicating cancer precursor lesions can be detected at similar levels in plasma compared with stool."

They concluded that their technique should be applicable to any other cancer-related gene mutations, although for the time being they will focus on providing further evidence of its utility in colorectal cancer screening.

"We would be very happy when our work could contribute to prevent cases of colon cancer in the near future by early detection of cancer precursors," Scholtka said. "In our previous work we have suggested a marker panel consisting of gene mutations in colon cancer-initiating genes. In a next step the WTB-HRM technique should be applied on this marker panel specifically selected for the detection of colon cancer precursors. The sensitivity and specificity of our technique should be validated in comparison with the standard screening method [of] colonoscopy in a representative number of patients."

Scholtka noted that the University of Potsdam holds a US patent, No. 7,833,757, describing a combination of PCR primers that would be especially suitable for use in a multitarget assay to detect colon cancer precursors and early cancerous stages, and that many of these same primers were used in the Cancer Prevention Research paper.

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