KRAS mutational test for metastatic colorectal cancer patients: not just a technical problem

We have with great interest read the review by Francesca Molinari and Milo Frattini: ‘KRAS mutational test for metastatic colorectal cancer patients: not just a technical problem’ [1]. Their thorough review discussing the pros and cons of our wobble-enhanced amplification refractory mutation system (WE-ARMS) method for detection of KRAS and BRAF mutations in comparison to other frequently used diagnostic methods was indeed to the point [2]. As pinpointed by the reviewers, “the authors state that each laboratory needs to establish the optimal cutoff, because it can depend on the type of sample, as well as on the method of DNA extraction”. We would like to comment on this statement, because we have gained some experience from testing various other tissues since the article was published in 2011.

The WE-ARMS method was applied to different types of cancers where the tissues had been subjected to various preprocessing and extraction methods (Table 1).

For all combinations stated in (Table 1) we used the same cutoff values as previously published without alterations. There were no borderline cases where mutation calling was questioned or the amplification curves were ambiguous. Optimizing the cutoff may be necessary in other circumstances, but our data support that the method is highly stable across tissue types, preprocessing steps and extraction methods.

In addition, when previously validating the WE-ARMS method we included a series of 49 formalin-fixed, paraffin-embedded tissues from metastatic colorectal cancer patients treated with anti-EGF receptor monoclonal antibodies. The tissue blocks were collected from 19 hospitals throughout the Nordic countries. The obtained results, published in our article, proved that this did not have a significant impact on the performance of the method [2].

In addition, we used the WE-ARMS method for BRAF mutational analysis of formalin-fixed, paraffin-embedded tissues from metastatic colorectal cancer patients in the NORDIC-VII study [3].

In our experience from analyzing various tissues preprocessed and extracted with different protocols, the WE-ARMS method does reflect a higher robustness than initially anticipated.

Table 1. Additional cancer tissues tested with the wobble-enhanced amplification refractory mutation system method.

Type of cancer	Tissue collection and storage	Extraction method	Cases (n)	Tumor cells preidentified
Pancreas	Formalin-fixed, paraffin-embedded	QIAamp^® DNA Micro, scraped tumor areas from sections on slides	95	Yes
Pancreas	Fresh frozen in liquid nitrogen and stored at -80°C	Sections homogenized in lysis buffer, extracted on Maxwell^® 16, Promega	5	Yes
Lung	Fresh frozen in liquid nitrogen and stored at -80°C	Trizol-based extraction of ‘tissue lumps’	241	No
Head and neck	Fixed in ethanol and stored at -80°C	‘Tissue lumps’ air-dried, cut with scissors in lysis buffer, homogenized, extracted on Maxwell^® 16, Promega	32	No

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.