Coamplification At Lower Denaturation temperature-PCR Research Articles

The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

A sensitive and convenient method for the detection of epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs) would be desirable to guide treatment strategy. Consequently, studies have focused on sensitive characterization of EGFR T790M mutation. Herein, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and pyrosequencing were combined (COLDPCR/ pyrosequencing) for detecting EGFR T790M mutation. Evaluation of mutation-containing dilutions revealed that the sensitivities of COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing assays for the detection of the T790M mutation were 0.1 and 5%, respectively, indicating a 50-fold increase in sensitivity. When the T790M mutation in 20 clinical NSCLC samples who had relapsed under firstgeneration EGFR TKI were further determined using COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing, the detection rates were 35% (7/20) and 25% (5/20), respectively. All patients who were positive for the T790M mutation with conventional PCR/pyrosequencing were also found to be positive with COLD-PCR/pyrosequencing. The discordant cases were 2 samples with no T790M mutation detected with conventional PCR/pyrosequencing, but which were positive with COLD-PCR/pyrosequencing. COLD-PCR/pyrosequencing is a sensitive and cost-effective tool for detecting the T790M mutation which will permit an improvement of therapeutic management.

Establishment of Coamplification at Lower Denaturation Temperature PCR/Fluorescence Melting Curve Analysis for Quantitative Detection of Hepatitis B Virus DNA, Genotype, and Reverse Transcriptase Mutation and Its Application in Diagnosis of Chronic Hepatitis B

Dynamic and real-time hepatitis B virus (HBV) DNA, genotype, and reverse transcriptase mutation analysis plays an important role in diagnosing and monitoring chronic hepatitis B (CHB) and in assessing the therapeutic response. We established a highly sensitive coamplification at lower denaturation temperature PCR (COLD-PCR) coupled with probe-based fluorescence melting curve analysis (FMCA) for precision diagnosis of CHB patients. The imprecision with %CV and detection limit of HBV DNA detected by COLD-PCR/FMCA were 2.58% to 4.42% and 500 IU/mL, respectively. For mutation, the imprecision and detection limit were 3.35% to 6.49% and 1%, respectively. Compared with Sanger sequencing, the coincidence rates of genotype and mutation were 96.0% and 82.5%, respectively, whereas the inconsistent data resulted from a low proportion (<20%) of mixed genotypes or mixed mutations. The mutation ratio in HBV infection patients was as follows: hepatitis B e antigen (HBeAg)-positive infection (0/0.0%)<HBeAg-negative infection (16/4.5%)<HBeAg-positive hepatitis (30/5.5%)<HBeAg-negative hepatitis (36/6.5%). In patients with entecavir therapy, the proportion of mutation at baseline or week 4 in virologic response (VR) group was <4%, whereas in the partial VR group, it was mostly≥4%. COLD-PCR/FMCA provides a novel tool with high sensitivity, convenience, and practicability for the simultaneous quantification of HBV DNA, genotype, and mutation. It might be used for distinguishing the different phases of HBV infection and predicting VR of CHB patients.

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies.

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR)for the sensitive detection of disease-related hypermethylation.

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Abstract 1390: High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR

Abstract Introduction: The EGFR Exon 20 T790M mutation confers resistance to the first generation tyrosine kinase inhibitors (TKI), such as Tarceva®. A new generation of treatments, including AZD9291, targets the T790M mutation thus allowing a new approach to cancer therapy for NSCLC patients. Recently EGFR Exon 20 C797S mutations (c.2389T&amp;gt;A and c.2390G&amp;gt;C) have been identified as resistance mutations to the third generation TKI AZD9291. High sensitivity detection of these mutations, as well as the T790M mutation, in patients with acquired resistance to third generation TKI is an important requirement for best practice NSCLC treatment. Materials and Methods: ICE COLD-PCR (Improved &amp; Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide (RS-oligo) complementary to the wild-type sequence. Due to the inherent sequence characteristics of the region between the codons of interest (790 and 797), a RS-oligo focusing on the C797S was developed and is independent of the RS-oligo for T790M, and thus tiling across EGFR Exon 20 can be performed. The RS-oligo in this assay was designed to prevent PCR amplification of wild-type sequences while allowing exponential amplification of any mutations including C797S within the RS-oligo binding region. Serial dilutions of spiked gBlocks® DNA containing C797S mutations mixed with K562 DNA and samples extracted from plasma were amplified with ICE COLD-PCR followed by Sanger sequencing analysis. Result: For mutations c.2389T&amp;gt;A and c.2390G&amp;gt;C, limits of detection as low as 0.05% were achieved. Forty DNA samples extracted from plasma were also tested. These indicated that the assay is suitable for fragmented samples with low quantities of input DNA such as liquid biopsies. Conclusion: The pre-amplification PCR product covers both codons 790 and 797. This allows ICE COLD-PCR to be used for the analysis of both T790M and C797S using the same liquid biopsy sample aliquot. This new ICE COLD-PCR method to detect both C797S mutations c.2389T&amp;gt;A and c.2390G&amp;gt;C may provide a highly sensitive, simple and cost effective assay for monitoring acquired resistance to AZD9291 in the liquid biopsies obtained from patients and clinical trials. Coupled with our existing highly sensitive assay for T790M, ICE COLD-PCR offers a highly sensitive method to determine the emergence of resistance mutations, thus potentially contributing to therapeutic optimization. Citation Format: Grant Wu, Sheena Jensen, Karissa Scott, Erin Montagne, Jason Stoddard, Phil Krzycki, Courtney Schweikart, Benjamin L. Legendre, Philip Eastlake, Katherine A. Richardson. High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1390.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma

BackgroundUntil now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through...

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

COLD-PCR protocols to enrich KRAS codon 61 and 146 mutations in heterogeneous tumor samples.

e22125 Background: About 40% of colorectal cancer patients show mutations on codon 12-13 of KRAS but other, less frequent and mutually exclusive mutations, were described. In Italy, treatments with Cetuximab or Panitumumab for metastatic colorectal cancer are granted only for patients with no mutations on RAS genes and analysis of codon 12,13,59,61,117,146 of KRAS and NRAS are requested before the treatment scheduling. Principal limitation of conventional PCR-based methods for mutation analysis is the inability to selectively amplify low percentages of somatic variants in an elevated background of wild-type sequences. CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) is a simple approach of PCR that selectively enriches minority alleles in a mixture of wild-type and mutant sequences. This method requires the definition of a Critical Temperature (Tc) for each Dna sequence, lower than the melting temperature, at which mismatched Dna heteroduplex (WT-mutated alleles) are preferentially denatured, promoting selective amplification of mutant sequences. Minor allele enrichment by COLD-PCR can be performed in association to different methods of variants detection: Sanger Sequencing, HRM analysis, Pyrosequencing Methods: Comparison of theoretical sensitivity achievable after PCR or COLD-PCR amplification was carried out analyzing serial dilution of mutated cell lines DNA into WT DNA (from 50% to 0.25%). To optimize the enrichment of minority alleles we developed a Full COLD-PCR protocol to analyze the Tm-increasing mutation (183 A>C) on KRAS codon 61 and a Fast COLD-PCR protocol to analyze the Tm-reducing mutation (436 G>A) on KRAS codon 146. Critical temperature was defined experimentally. Results: In KRAS codon 61 and 146 analysis, minimum detectable percentage of mutated allele in WT background was about 10% using conventional PCR followed by both Sanger sequencing and HRM. Conversely limit of sensitivity reached 0.5% using both detection methods after COLD-PCR Conclusions: Our experiments proved as COLD-PCR could be a rapid and economic approach, adaptable to investigate other somatic variants, to enrich very low percentages of mutant alleles in very heterogeneous tumor samples.

A noninvasive assay for monitoring renal allograft status

Transplant rejection is a serious complication, sometimes threatening life of the patient. Although recent development of the new generation of immunosuppressive drugs reduced the incidence of acute rejection in kidney transplantation, the absence of noninvasive biomarkers of the rejection does not allow often the optimization of a prompt antirejection therapy. Serum creatinine is the most widely used marker for allograft function, however, it is not sensitive and specific enough to detect acute rejection. Other biomarkers are even less valuable for this purpose. Histological examination of renal allograft biopsy still remains the golden standard for diagnosing acute renal allograft rejection. Therefore, there is a high demand for reliable biomarkers for noninvasive monitoring of renal allograft status. Examination of urine in renal transplant recipients provides a logical and readily accessible approach for this monitoring. The high potency biomarkers for kidney allograft monitoring are fragments of DNA in recipient urine that originated from renal allograft cells. Because of the difference in the genetic origin these DNA can be distinguished from recipient DNA. Quantitative analysis of donor’s DNA, derived from cells of renal allograft, in recipient’s urine might be a reliable predictive tool for the kidney transplant rejection. We developed an assay to quantitate donor DNA content in recipient urine. Application of the technique—coamplification at lower denaturation temperature-PCR (COLD-PCR) increased the abundance of donor DNA that usually presents in recipient urine in quantities that are out of the detection range. This assay has a potential for routine application in clinical practice after statistical validation and additional modifications.

The “COLD-PCR Approach” for Early and Cost-Effective Detection of Tyrosine Kinase Inhibitor Resistance Mutations in EGFR-Positive Non–Small Cell Lung Cancer

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report

Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

Reply to the article entitled “Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report” by Galbiati et al., Clin Chem Lab Med 2014;52(4):505–9

Article Reply to the article entitled “Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report” by Galbiati et al., Clin Chem Lab Med 2014;52(4):505–9 was published on July 1, 2014 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 52, issue 7).

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

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 BRAFV600E 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.

COLD-PCR and Innovative Microarray Substrates for Detecting and Genotyping MPL Exon 10 W515 Substitutions

Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.

Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment

Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

COLD-PCR Enrichment of Rare Cancer Mutations prior to Targeted Amplicon Resequencing

Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing. We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000. In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent. As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.

Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR

Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis.

Use of BLOCker sequencing (blocking oligonucleotide cycle sequencing) after ice COLD-PCR for detection of K-RAS mutations.

e13547 Background: The epidermal growth factor receptor (EGFR) antagonists, cetuximab and panitumumab, can be effective in colorectal cancer treatment. However, activating K-RAS mutations in codons 12 and 13 are associated with a poor EGFR antagonist response. As these mutations are seen in ~40% of CRC, high sensitivity assays for detection of these mutations will aid in patient treatment and disease monitoring. Methods: Ice COLD-PCR (Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) uses a reference sequence oligonucleotide (RS-oligo) to enhance detection of all low level mutations during PCR. The RS-oligo is complementary to one wild-type strand, so only one wild-type strand but both mutant DNA strands are amplified. This results in linear WT sequence amplification but exponential mutant sequence amplification. Ice COLD-PCR methods used in these studies can detect 0.05 – 0.01% K-RAS mutant DNA. For low level mutation confirmation we added BLOCker-oligos to sequencing reactions to block wild-type and allow mutant DNA sequencing (BLOCker-Sequencing). For blocking to occur, additional hybridization and denaturing steps are added to the cycle sequencing program. This insures that the BLOCker-oligo:WT DNA remains intact but the BLOCker-oligo:Mutant DNA is denatured. A sequencing primer overlapping the BLOCker-oligo 5’ end anneals to the mutant strand that is subsequently extended. Results: BLOCker sequencing was used to confirm presence or absence of low level K-RAS mutations after Ice COLD-PCR. When a K-RAS mutation is present at 0.05%-0.01%, the mutant peak may appear as baseline noise. When BLOCker Sequencing of these samples is performed, the presence of a mutation at this low level is enhanced in the BLOCker Sequencing electropherogram. There is no enhancement if the sample does not contain a mutation. Conclusions: Following Ice COLD-PCR amplification, BLOCker sequencing was able to confirm the presence or absence of very low levels of K-RAS mutations in the starting samples. BLOCker Sequencing is a novel method to confirm low level mutation presence when there is a uncertain DNA sequencing electropherogram result following Sanger sequencing.

COLD-PCR: improving the sensitivity of molecular diagnostics assays

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.