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KRAS Diagnostic Testing

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Vol. 20 • Issue 5 • Page 12

The Molecular Edge

Epidermal growth factor receptor (EGFR) signaling is involved in the initiation, maintenance and progression of multiple human malignancies, including pancreatic, non-small cell lung, colorectal and thyroid carcinomas. EGFR signaling activates down-stream proteins such as KRAS, BRAF, mTOR and PIK3CA, promoting malignant transformation, angiogenesis, cell growth, migration and survival. Recently, anti-EGFR therapies have been developed for locally advanced or metastatic colorectal and non-small cell lung cancers that have failed prior chemotherapeutic regimens. Specifically, the anti-EGFR antibodies panitumumab and cetuximab are employed in treating colorectal carcinoma and EGFR tyrosine kinase inhibitors are used for non-small cell lung cancers. Numerous studies have demonstrated that activating KRAS gene mutations are associated with anti-EGFR therapy resistance and the National Comprehensive Cancer Network recommends all patients with colorectal or non-small cell lung cancer being considered for anti-EGFR therapy be tested for KRAS mutations. Most activating KRAS mutations are point mutations found in codons 12 and 13 of exon two, with a lower number in exon 61. Presently, KRAS testing usually involves the analysis of eight to 12 different point mutations.

Clinical testing for CRC and non-small cell lung cancers is commonly performed on frozen, formalin-fixed or formalin-fixed paraffin-embedded tissue blocks. Testing of KRAS mutations from formalin-fixed tumors can be challenging, as the DNA can be fragmented, cross-linked to other biomolecules, may contain PCR inhibitors and may have formalin-fixation introduced DNA sequence alterations. Additionally, benign stromal cells within a tumor may significantly dilute mutant KRAS sequences. For these reasons the highest sensitivity and specificity are often required for the successful clinical specimen analysis. Often macro/microdissection is initially employed to maximize the amount of tumor in the sample prior to DNA purification.

Multiple methods are used for KRAS testing, including sequencing, high-resolution melting analysis, array/strip analyses and allele-specific PCR. All these techniques have been successfully applied to clinical KRAS testing and each has its unique features.

Sequencing

KRAS sequencing is usually performed by Sanger or pyrosequencing, with the former often considered the testing gold standard. The detection limit of each technique is ~15% and 6%; this is acceptable for most clinical specimens but low for those with low tumor cell numbers such as post-chemotherapy treated tumor beds. For each method the genomic DNA surrounding KRAS mutations are PCR amplified and each base is interrogated following the addition of a primer, nucleotides and DNA polymerase. In the Sanger method, labeled DNA synthesis-terminating nucleotides are added to the DNA synthesizing reaction, creating a "DNA ladder" of different molecular weights separated by size and analyzed by the specific fluorolabel they carry-one for each nucleotide. From this information the specific DNA sequence can be identified.

In pyrosequencing, limited amounts of each of the four nucleotides are added one at a time. Upon nucleotide incorporation visible light is chemically produced by luciferase. The sequence is determined from the correlation of light appearing and nucleotide type introduced. Sequencing has the advantage of interrogating every base for a possible mutation. Sequence testing is reliable, has a good turnaround time and high specificity, but can have low sensitivity and requires the use of expensive lab equipment. Sanger and pyrosequencing require different instruments, with different costs, system requirements and bioinformatic analysis programs. Roche Diagnostics, Qiagen and Beckman Coulter sell Sanger and pyrosequencers that can be used for KRAS mutation analysis.

High-resolution Melting Analysis

Like sequencing, this method begins by PCR amplifying genomic DNA surrounding known KRAS mutations. The amplicons are renatured and slowly heated in the presence of a fluorophore that emits strongly when bound to the dsDNA, but not ssDNA. Mutated amplicons will denature at different temperatures than wild type amplicons, as they have different base pairs and altered thermostability. This method allows the rapid comparison of many samples, lending itself to high-throughput systems, has a relatively low cost and can be done in a single closed system, lowering the risk of false results due to cross contamination. It does not identify the specific bases mutated and can give a high number of false-positive results. For this reason, it is recommended that all results be confirmed by another testing method. Most commercial KRAS testing methods do not use this technique.

Allele-specific PCR

This technique uses multiple PCR primers specifically designed to amplify either wild type or one of the KRAS mutations. The 3' end of a mutation-specific primer is matched to a specific KRAS mutation. Amplification will only occur where the base pairs match, as the Taq polymerase initiates PCR poorly from mismatched bases. Amplification is detected when the Taq 5'exonucleas activity degrades a bound DNA probe carrying an attached emitter-quencher pair. Once freed the emitter fluoresces, indicating that amplification is occurring. This technique has high sensitivity, detecting as little as 1% or lower mutant DNA within a sample, has high specificity, moderate cost and a rapid turnaround time. Since the Taq enzyme will initiate amplification from mismatched bases at a low rate, a sample is only considered mutation positive when the number of PCR amplification (Ct) cycles is 35 or less. Entrogen offers an allele-specific PCR kit for clinical KRAS and BRAF mutation analysis.

Array/Strip Analysis

In this technique, PCR-amplified KRAS genomic DNA or cDNA is labeled and hybridized to specific mutant and wild type KRAS sequences attached to a solid support, such as a glass slide or nitrocellulose membrane strip. Following hybridization and washing steps, automated analysis is used to measure the specific hybridization pattern(s) seen and determine what specific KRAS mutations are present within a sample. Although not commonly employed, the array/strip technique is sensitive and specific, detecting 0.1% to 1% mutant KRAS sequences diluted in wild type DNA. The cost and turnaround time of array compare to other methods; in the future, this testing method will probably be increasingly used. TrimGen, NLM, Oasis and Vienna Lab Diagnostics offer kits for array KRAS testing.

The identification of an activating KRAS mutation indicates that anti-EGFR therapies are unlikely to benefit individuals with advanced colorectal or non-small cell lung cancers. Additionally, these therapies, such as panitumumab and cetuximab, are expensive (~$10,000/month) and do not benefit individuals with KRAS mutations. Thus, KRAS testing lowers medical costs and reduces patient morbidity. There is no clinical need to identify the specific KRAS mutation present for patient management, although this may change, as one study (published in JAMA, 2010;304:1812-20) indicates that individuals with the G13D KRAS mutation may still respond to cetuximab.

Dr. Shackelford is with the Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans.




     

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