G. Mike Makrigiorgos

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.

Bystander effect produced by radiolabeled tumor cells in vivo

The bystander effect, originating from cells irradiated in vitro, describes the biologic response(s) of surrounding cells not directly targeted by a radiation insult. To overcome the limitations of in vitro tissue culture models and determine whether a bystander effect that is initiated by the in vivo decay of a radionuclide can be demonstrated in an animal, the ability of 5-[125I]iodo-2′-deoxyuridine (125IUdR)-labeled tumor cells to exert a damaging effect on neighboring unlabeled tumor cells growing s.c. in nude mice has been investigated. When mice are injected with a mixture of human colon LS174T adenocarcinoma cells and LS174T cells prelabeled with lethal doses of DNA-incorporated 125I, a distinct inhibitory effect on the growth of s.c. tumor (derived from unlabeled cells) is observed. Because (i) the 125I present within the cells is DNA-bound, (ii) ≈99% of the electrons emitted by the decaying 125I atoms have a subcellular range (<0.5 μm), and (iii) the overall radiation dose deposited by radiolabeled cells in the unlabeled cells within the growing tumor is <10 cGy, we conclude that the results obtained are a consequence of a bystander effect that is generated in vivo by factor(s) present within and/or released from the 125IUdR-labeled cells. These in vivo findings significantly impact the current dogma for assessing the therapeutic potential of internally administered radionuclides. They also call for reevaluation of the approaches currently used for estimating the risks to individuals and populations inadvertently exposed internally to radioactivity as well as to patients undergoing routine diagnostic nuclear medical procedures.

A PCR-based amplification method retaining the quantitative difference between two complex genomes

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.

PCR-Based Methods for the Enrichment of Minority Alleles and Mutations

BACKGROUND The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze. CONTENT Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change. SUMMARY This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.

Localized Dose Enhancement to Tumor Blood Vessel Endothelial Cells via Megavoltage X-rays and Targeted Gold Nanoparticles: New Potential for External Beam Radiotherapy

PURPOSE Tumor endothelial cell damage during radiation therapy may contribute significantly to tumor eradication and treatment efficacy. Gold nanoparticles (AuNPs) delivered preferentially to the walls of tumor blood vessels produce low-energy, short-range photoelectrons during external beam radiotherapy, boosting dose to the tumor microvasculature. In this study dosimetry at the single-cell level is used to estimate the anticipated AuNP-mediated dose enhancement to tumor endothelial cells during 6-MV X-ray irradiation. METHODS AND MATERIALS Endothelial cells are modeled as thin slabs with 100-nm-diameter AuNPs attached within the blood vessel. The number of photoelectrons emitted per AuNP per gray of X-rays is computed at multiple points along the external beam central axis by use of a Monte Carlo-generated energy fluence spectrum. The energy deposited from AuNP emissions to the endothelium is calculated based on an analytic method incorporating the energy-loss formula of Cole. The endothelial dose enhancement factor (EDEF) is the ratio of the overall (externally plus internally generated) dose to endothelial cells in the presence of AuNPs to the dose without AuNPs (from the external beam only). RESULTS At 20-cm depth, the EDEF is 1.7 (70% dose increase) for an intravascular AuNP concentration of 30 mg/g. Most of this dose enhancement arises from the low-energy (approximately 100 keV) portion of the linear accelerator X-ray spectrum. Furthermore, for AuNP concentrations ranging from 7 to 140 mg/g, EDEF values of 1.2 to 4.4 (20-340% dose increase) are calculated. CONCLUSIONS In contrast to calculations assuming that AuNPs distributed homogeneously throughout the target volume (macrodosimetry), our cellular microdosimetry calculations predict a major dose enhancement to tumor microvasculature from conventional linear accelerator X-rays. This effect may enable the delivery of ablative therapeutic doses to these sensitive microstructures while maintaining established dose constraints for the organs at risk.

Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.

Reproducible and inexpensive probe preparation for oligonucleotide arrays

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

COLD-PCR–Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations

BACKGROUND Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature-PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples. METHODS Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6-8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced. RESULTS Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons. CONCLUSIONS COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.

Targeted radiotherapy with gold nanoparticles: current status and future perspectives

Radiation therapy (RT) is the treatment of cancer and other diseases with ionizing radiation. The ultimate goal of RT is to destroy all the disease cells while sparing healthy tissue. Towards this goal, RT has advanced significantly over the past few decades in part due to new technologies including: multileaf collimator-assisted modulation of radiation beams, improved computer-assisted inverse treatment planning, image guidance, robotics with more precision, better motion management strategies, stereotactic treatments and hypofractionation. With recent advances in nanotechnology, targeted RT with gold nanoparticles (GNPs) is actively being investigated as a means to further increase the RT therapeutic ratio. In this review, we summarize the current status of research and development towards the use of GNPs to enhance RT. We highlight the promising emerging modalities for targeted RT with GNPs and the corresponding preclinical evidence supporting such promise towards potential clinical translation. Future prospects and perspectives are discussed.

In vitro radiosensitization by gold nanoparticles during continuous low-dose-rate gamma irradiation with I-125 brachytherapy seeds

UNLABELLED This communication reports the first experimental evidence of gold nanoparticle (AuNP) radiosensitization during continuous low-dose-rate (LDR) gamma irradiation with low-energy brachytherapy sources. HeLa cell cultures incubated with and without AuNP were irradiated with an I-125 seed plaque designed to produce a relatively homogeneous dose distribution in the plane of the cell culture slide. Four sets of irradiation experiments were conducted at low-dose rates ranging from 2.1 to 4.5cGy/h. Residual γH2AX was measured 24h after irradiation and used to compare radiation damage to the cells with and without AuNP. The data demonstrate that the biological effect when irradiating in the presence of 0.2mg/ml concentration of AuNP is about 70%-130% greater than without AuNP. Meanwhile, without radiation, the AuNP showed minimal effect on the cancer cells. These findings provide in vitro evidence that AuNP may be employed as radiosensitizers during continuous LDR brachytherapy. FROM THE CLINICAL EDITOR In this basic science paper, the application of gold nanoparticles as radiosensitizing agents for low dose rate gamma radiation therapy is discussed, demonstrating efficacy in cell culture models.