Lilin Wang

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non-Small Cell Lung Cancer

Purpose: Tumors from 50% of epidermal growth factor receptor (EGFR) mutant non–small cell lung cancer patients that develop resistance to gefitinib or erlotinib will contain a secondary EGFR T790M mutation. As most patients do not undergo repeated tumor biopsies we evaluated whether EGFR T790M could be detected using plasma DNA. Experimental Design: DNA from plasma of 54 patients with known clinical response to gefitinib or erlotinib was extracted and used to detect both EGFR-activating and EGFR T790M mutations. Forty-three (80%) of patients had tumor EGFR sequencing (EGFR mutant/wild type: 30/13) and seven patients also had EGFR T790M gefitinib/erlotinib-resistant tumors. EGFR mutations were detected using two methods, the Scorpion Amplification Refractory Mutation System and the WAVE/Surveyor, combined with whole genome amplification. Results: Both EGFR-activating and EGFR T790M were identified in 70% of patients with known tumor EGFR-activating (21 of 30) or T790M (5 of 7) mutations. EGFR T790M was identified from plasma DNA in 54% (15 of 28) of patients with prior clinical response to gefitinib/erlotinib, 29% (4 of 14) with prior stable disease, and in 0% (0 of 12) that had primary progressive disease or were untreated with gefitinib/erlotinib. Conclusions:EGFR T790M can be detected using plasma DNA from gefitinib- or erlotinib-resistant patients. This noninvasive method may aid in monitoring drug resistance and in directing the course of subsequent therapy.

Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR

BACKGROUND DNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. METHODS Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. RESULTS COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. CONCLUSIONS The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Preferential amplification of apoptotic DNA from plasma: potential for enhancing detection of minor DNA alterations in circulating DNA.

Tumors release genomic DNA into the circulation of cancer patients after cellular necrosis and apoptosis. Isolation of the apoptotic fraction of plasma-circulating DNA can enhance detection of low-level mutations that can serve as tumor biomarkers (1). Because the amount of DNA circulating in the plasma of cancer patients is low, on the order of a few nanograms per milliliter of blood, the number of genes that can be examined for tumor-specific alterations is limited, a situation that reduces biomarker sensitivity. We recently applied whole-genome amplification of plasma-circulating DNA to increase the number of targets that can be analyzed from each sample, thus potentially increasing biomarker sensitivity(2). This approach yields highly-expanded DNA amounts for performing genetic screening; however, there is no preferential enrichment of smaller sized DNA fragments. We report a new method for whole-genome amplification of plasma-circulating DNA, based on ligation-mediated PCR of blunted DNA fragments (BLM-PCR)1 , which results in preferential amplification of smaller size, apoptotic DNA fragments. Plasma-circulating DNA was extracted from blood obtained from radiation therapy patients after the patients gave informed consent and the study received institutional review board approval. Within 2–3 h of collection, whole blood was centrifuged at 2000 g for 15–30 min, plasma was separated, and plasma-circulating DNA was purified by use of a QIAamp™ MinElute Virus Spin Kit (Qiagen) and quantified via Taqman real-time-PCR. To test for v-Ki-ras2 Kirsten …

High-throughput scanning of breast tumor surgical specimens for low-level mutations.

Abstract Large numbers of mutations are postulated to occur as early events in carcinogenesis. For certain types of human tumors (mutator phenotypes) these mutations can be a driving force in generating clonogenic, causative genetic changes leading to multistage carcinogenesis. These low-level mutational events are highly significant due to their potential use as molecular markers for early identification of genomic instability that can lead to cancer and to their potential influence on the ability of tumors to resist drug treatment and/or metastasize. Detecting the presence and diversity of such genetic changes in human tumors is desirable due to their potential prognostic value. However, identification of these low-frequency genetic changes is difficult, since most mutations exist at mutant/wild-type ratios of <10 −3. We recently developed inverse PCR-based amplified restriction fragment-length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing HPLC to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP we previously demonstrated low-level mutations (mutation frequency <10 −3) in human colon cancer cells that harbor mismatch repair deficiency and in sporadic colon cancer surgical specimens. In the present work we investigated whether low-level mutations are also present in sporadic breast cancer surgical specimens. Using iFLP we identified widespread low-level mutations in two out of ten surgical specimens examined (20%). Examination of the microsatellite instability status of these samples demonstrated that the samples are stable (MSI-S). We conclude that low-level mutations are less frequent in breast cancer than in colon cancer; however, single nucleotide instability that generates such mutations may still be present in a fraction of breast cancers.

Abstract 2090: Mutational analysis in circulating tumor cells (CTC):ScreenCell MB Filtration Unit and ICE COLD-PCR

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The aim of this study was to validate the use of the ScreenCell® MB Filtration Unit for detection of mutations in Circulating Tumor Cells. This device allows physicians to easily isolate any CTCs using a simple blood draw which can be performed at a local doctors office. This is especially important when trying to analyze CTCs in elderly patient populations and in regions where the large medical centers are far away. Once isolated, the filter containing the CTCs can be sent to a laboratory for DNA isolation and the mutation analysis. Mutational analysis using ICE COLD-PCR can then be performed without the use of expensive or specialized equipment. The mutation was characterized using standard Sanger DNA sequencing with a detection limit of 0.5 - 0.1% mutant sequence in the wild-type background. Results will be presented from a mock experiment where PC9 cells (EGFR exon 19 deletion: p.E746\_A750del) or H1975 cells (EGFR exon 21: p.L858R) were spiked into blood and then these mock CTCs were collected on the ScreenCell Molecular Filter Device. Following DNA isolation, the mutations were easily detected at the lowest number of cells spiked in to the blood samples (10 cells). Additional data from CTCs collected from NSCLC patients detected the L858R point mutation, an exon 19 E746\_A750del mutation, and an exon 19 E746_S752delinsV. Mutations in these same samples could not be detected in DNA sequencing following standard PCR amplification. These data show that the ScreenCell MB Filtration Unit coupled to mutational analysis using ICE COLD-PCR can be an easy, cost-effective method for collection and analysis of CTCs in patient blood samples. Moreover, the device is simple enough to be used in any doctors office. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2090. doi:1538-7445.AM2012-2090