A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

<div><p>Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAF^V600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.</p> </div

Relative fluorescence intensity for detecting the sensitivity of the system.

<p>Detection limit of the G13D mutation (A) and of the V600E BRAF mutation (B). wt: wild-type control samples; het1, het2 and het3: heterozygous control samples; numbers from 1 to 10: serial dilutions, 1 = 6%, 2 = 3%, 3 = 1,6%, 4 = 0,8%, 5 = 0.4%, 6 = 0.2%, 7 = 0.1%, 8 = 0.05%, 9 = 0.02%, 10 = 0.01% respectively. Bars are the average of the intensity of the 6 replicates of each sample. The error bars are the standard deviations of the fluorescence intensity of each sample.</p

Microarray image for the analysis of the G12C KRAS mutation samples.

<p>(A) Cy5 fluorescence signal corresponding to the mutated allele. (B) spotting scheme. wt: wild-type control samples; het1, het2 and het3: heterozygous control samples for G12A, G12C, G12D, G12R, G12S, G13D, G12V KRAS mutations; numbers from 1 to 60: sixty different solid tumour DNA samples. Grey markers represent the samples positive for G12C mutation; light grey squares represent an amino-modified oligonucleotide labelled with Cy3 used as reference spots. (C) HRM analysis of sample number 30 (number 2) compared to melting profiles of control samples (wild-type reference = number 1; mutated reference = number 3) after amplification by COLD-PCR: the melting behaviour is suggestive for the presence of mutated DNA in the sample. (D) direct sequencing analysis of sample number 30 submitted to COLD-PCR amplification protocol: the electropherogram confirms the presence of mutated DNA (G12C) in the sample.</p

Assay scheme.

<p>Hybridization steps: 1) stabilizer oligonucleotide to open the secondary structure of the PCR fragment; 2) The “universal reporter mix”. The mix contains the wild-type and mutant reporters. Each reporter is prolonged by a tail complementary to the labeled universal oligonucleotide. Cyanine 3- (Cy3) and Cyanine 5- (Cy5) labeled universal oligonucleotide anneal to wild-type and mutant reporters, respectively.</p

Sequences of reporters and stabilizer oligonucleotides.

*†<p>Universal reporter formats which hybridize to the universal probes (in small letter); * wild-type sequence-specific tail; ^† mutant sequence-specific tail.</p><p>Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference GenBank sequence (NM_033360.2). The initiation codon is codon 1.</p

Microarray image for genotyping the G12R KRAS mutation.

<p>(A) microarray scanning of the Cy3 fluorescence signal corresponding to the wild-type allele. Spots in column 1,2,3,4 represent amino-modified oligonucleotide labelled with Cy3 used as reference spots. (B) scanning of the Cy5 fluorescence signal corresponding to the mutated allele. (C) microarray spotting scheme. wt: wild-type control samples; het1, het2 and het3: heterozygous control samples for G12A, G12C, G12R, G12S, G12V, G13D; G12D KRAS mutations; light grey squares represent amino-modified oligonucleotide labelled with Cy3 used as reference spots. (D) normalized relative fluorescence intensity after hybridization of known control samples with the reporters complementary to the G12R variation. Bars are the average of the intensity of the 6 replicates of each sample. The error bars are the standard deviations of the fluorescence intensity of each sample.</p

Microarray image for genotyping the V600E BRAF mutation.

<p>(A) Cy3 fluorescence signal corresponding to the wild-type allele. Spots in column 1,2,3,4 represent amino-modified oligonucleotide labelled with Cy3 used as reference spots. (B) Cy5 fluorescence signal corresponding to the mutated allele. (C) microarray spotting scheme. wt: wild-type control samples; het1, het2 and het3: heterozygous control samples; mut: homozygous mutant control sample; light grey squares represent amino-modified oligonucleotide labelled with Cy3 used as reference spots. (D) normalized relative fluorescence intensity after hybridization of known control samples with the reporters complementary to the V600E BRAF variation. Bars are the average of the intensity of the 6 replicates of each sample. The error bars are the standard deviations of the fluorescence intensity of each sample.</p

Microarray image for the analysis of the V600E BRAF mutation samples.

<p>(A) Cy3 fluorescence signal corresponding to the wild-type allele. Spots in column 1,2,3,4 represent amino-modified oligonucleotide labelled with Cy3 used as reference spots. (B) Cy5 fluorescence signal corresponding to the mutated allele. (C) spotting scheme. wt: wild-type control samples; BRAF het1, het2 and het3: heterozygous control samples; mut: homozygous mutant control sample; numbers from 1 to 60: sixty different solid tumour samples. Grey markers represent the samples positive for BRAF mutation; light grey squares represent an amino-modified oligonucleotide labelled with Cy3 used as reference spots.</p

Molecular features of all patients enrolled by HRM-direct sequencing, and microarray.

*<p>mutation detected only by HRM and sequencing; by microarray analysis, the sample was identified as wild-type since no specific reporter was designed for the mutation.</p>†<p>mutation detected by COLD-PCR and sequencing, non detected by conventional PCR.</p

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics