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The Journal of Molecular Diagnostics : JMD logoLink to The Journal of Molecular Diagnostics : JMD
. 2016 Jul;18(4):546–553. doi: 10.1016/j.jmoldx.2016.02.004

Hybridization-Induced Aggregation Technology for Practical Clinical Testing

KRAS Mutation Detection in Lung and Colorectal Tumors

Hillary S Sloane ∗, James P Landers ∗,†,‡, Kimberly A Kelly §,¶,∗
PMCID: PMC4929403  PMID: 27289420

Abstract

KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA—a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways.

Advances in understanding the genetic basis of cancer have heralded a new era of clinical oncology, and with it, the prospect of a patient-centered model of cancer care. Specifically, the identification and functional analysis of tumor-specific genetic alterations have opened exciting opportunities for exploiting genetic mutations as predictors of therapeutic response and for guiding, in unprecedented ways, a more effective treatment regimen.1 A compelling example has been the emergence of KRAS mutations as powerful predictive biomarkers of treatment sensitivity in patients with colorectal cancer and non–small cell lung cancer.2

Approximately 30% to 40% of colorectal cancer tumors and 20% of non–small cell lung cancer tumors are known to harbor KRAS point mutations, occurring almost always in codon 12 or 13.3 These mutations render the Kirsten ras (KRas) protein constitutively GTP bound and active, and consequently lead to stimulus-independent, persistent activation of downstream effectors to promote cell proliferation, survival, and metastasis.4 Thus, KRAS mutations provide a mechanism for bypassing the antitumor effect of therapeutic strategies directed to the epidermal growth factor receptor, an upstream receptor tyrosine kinase in the KRas pathway. Indeed, numerous clinical studies have demonstrated that response to anti–epidermal growth factor receptor therapies (including the monoclonal antibody therapies cetuximab and panitumumab as well as the tyrosine kinase inhibitors gefitinib and erlotinib) is limited to patients harboring tumors with wild-type KRAS.2, 3, 5, 6, 7, 8, 9, 10, 11, 12 Given the overwhelming evidence, it is clear that the assessment of KRAS mutation status is an essential strategy for increasing the efficiency of treatment allocation in colorectal cancer and non–small cell lung cancer patients, to improve outcomes and reduce costs.13 Additionally, KRAS genotyping for population stratification will likely be important in accelerating the development of novel inhibitors of this key oncogenic pathway.14, 15

It has recently been advocated that KRAS testing be incorporated into routine practice for all colorectal cancer patients at the time of initial diagnosis (eg, reflex testing), as the current process of retrospective KRAS testing is cumbersome, time-consuming, and error prone.16 Although reflex testing appears an ideal scenario, its implementation raises questions about practicality. A number of technologies have been used for KRAS mutation analysis, including Sanger sequencing, pyrosequencing, and high-resolution melting analysis; however, it is unclear whether any current methodology could be applied in a low-maintenance (ie, operation by unskilled personnel and easy to interpret) and economically sound manner. Sanger sequencing is considered to be the gold standard, yet it requires the longest turnaround time and hands-on time of these methods.17 High-resolution melting analysis can be run considerably faster than other methods; however, it requires costly instrumentation18 and is often associated with unreliable results.19, 20 Pyrosequencing methods may have relatively lower costs per assay, but the instrumentation is the most costly to purchase and maintain.18, 21 The limitations associated with these methods provide a substantial barrier to their implementation in the routine clinical setting, where speed, simplicity, and cost-effectiveness are not only preferable but also essential.

In an effort to fulfill the unmet clinical demand for a methodology amenable to the requirements of routine testing, we recently introduced a novel approach for the detection of point mutations.22 The method (Figure 1) is an extension of hybridization-induced aggregation (HIA) technology, whereby the hybridization of a specific DNA target to a pair of oligonucleotide probes immobilized on the surface of microbeads, tethers the microbeads together and induces aggregation.23, 24 A digital image of the hybridization microwell is used for quantifying the extent of aggregation in terms of image saturation, which allows for a quantitative representation of hybridization efficiency. We exploit this phenomenon to differentiate target DNA sequences that differ by only a single nucleotide in a method we refer to as HIA for mutation detection (HIAMD). In a proof-of-principle demonstration of this technology, we proposed an assay to reveal KRAS mutations.22 The bead-bound oligonucleotide probes were designed with perfect complementarity to the wild-type KRAS gene segment; therefore, the presence of any of the common KRAS mutations (located in codons 12 and 13) in a target reduces the stability of the hybridization complex, resulting in decreased bead aggregation. This unique approach allows for the detection of any of the common KRAS mutations in a single-step, 2-minute assay, using only one set of oligonucleotide probes. The assay is performed at room temperature and uses simple, inexpensive instrumentation with a reusable plastic microwell chip that permits multiplexed analysis. The analytical parameters of the assay were optimized using synthetic oligonucleotide targets and RNA from cell lines. As our previous work suggested promise, we sought to establish the potential utility in a practical clinical context.22

Figure 1.

Figure 1

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Assay principles. Unpurified PCR product is added to a microwell containing probe-conjugated microbeads. A wild-type target hybridizes efficiently to both probes, inducing bead aggregation. Hybridization of a mutant target to the discriminating probe is unfavorable; therefore, the beads remain dispersed. An image of each microwell is obtained and processed to generate a saturation value corresponding to the extent of aggregation.

Here, we evaluate HIAMD as a potential technology for clinical testing and, specifically, for KRAS mutation screening in lung and colorectal cancers. Initial feasibility is established using lung and colorectal cancer cell lines. With the understanding that the analysis of patients' tumor samples requires mutation detection in a background of wild-type DNA, we investigate the utility of HIAMD in detecting KRAS mutations in low abundance. Finally, we apply the method for KRAS mutation analysis of 20 colorectal and lung patient tumors, comparing the results to those obtained from sequencing.

Materials and Methods

Oligonucleotides

Synthetic target sequences, biotinylated probe sequences, and PCR primers were synthesized by Eurofins MWG Operon (Huntsville, AL). All oligonucleotide sequences are listed in Table 1.

Table 1.

Oligonucleotide Sequences

Oligonucleotide Sequence
PCR primers
 HIAMD forward 5′-GACTGAATATAAACTTGTGGTAGTTGGA-3′
 HIAMD reverse 5′-CATATTCGTCCACAAAATGATTCTG-3′
 Sequencing forward 5′-GAGAATTCATGACTGAATATAAACTTGT-3′
 Sequencing reverse 5′-TCGAATTCCTCTATTGTTGGATCATATTCG-3′
Targets
 KRAS wild type 5′-GACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATG-3′
 KRAS mutant (c.34G>T) 5′-GACTGAATATAAACTTGTGGTAGTTGGAGCTTGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATG-3′
Probes
 Discriminating 5′-CTACGCCTCCAGCTCTTTTTT[Biotin-TEG∼Q]-3′
 Stabilizing 5′-[Biotin-TEG]TTTTTTCTGAATTAGCTGTATCGTCAAGGCACTC-3′
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Cell Lines

Cell lines, including SW-620 (colorectal carcinoma; c.35G>T, p.G12S), H2122 (non–small cell adenocarcinoma; c.34G>T, p.G12C), CaCo2 (colorectal carcinoma; wild-type), and H1975 (lung adenocarcinoma; wild-type), were purchased from the ATCC (Manassas, VA). cDNA was synthesized from cultured cells using the FastLane Cell cDNA Kit (Qiagen, Venlo, the Netherlands).

Tumor Samples

Twenty frozen surgical resections (10 lung and 10 colon) were obtained from the Department of Pathology through the Biorepository and Tissue Research Facility, University of Virginia (Charlottesville, VA). The samples were coded and collected under Institutional Review Board protocol. Sample characteristics are listed in Table 2. The samples were divided into two groups of 10 (each including 5 lung and 5 colon) for RNA isolation. Although genomic DNA could have also been used as the input nucleic acid, it has been suggested that RNA may be preferred over genomic DNA for mutation analysis of frozen tissue samples.25 The first group of samples (Patients 1, 3, 5, 10, 12, 13, and 18–20) was prepared using the mirVana miRNA Isolation Kit (Life Technologies, Carlsbad, CA) for total RNA isolation, and the second group (Patients 2, 6–9, 11, and 14–17) was prepared using the RNeasy Mini Kit (Qiagen). The concentration and purity of the RNA products were estimated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). RNA was then converted to cDNA using the QuantiTect Reverse-Transcription Kit (Qiagen). Approximately 1 μg of RNA was used in each 20-μL reverse-transcription reaction.

Table 2.

Patients' Tumor Samples

Patient no. Anatomic site Pathologic diagnosis Tumor differentiation % Tumor by cellularity Sequencing result HIAMD result
1 Colon Adenocarcinoma Moderate 60 wt wt
2 Lung Adenocarcinoma Well 90 wt wt
3 Colon Adenocarcinoma Well 40 wt wt
4 Colon Adenocarcinoma Poor 80 wt wt
5 Lung Squamous cell carcinoma Moderate 70 wt wt
6 Lung Adenocarcinoma Moderate 90 wt wt
7 Lung Squamous cell carcinoma Poor 90 wt wt
8 Colon Adenocarcinoma Poor 80 wt wt
9 Colon Adenocarcinoma Poor 80 wt wt
10 Colon Adenocarcinoma Moderate 80 wt wt
11 Colon Adenocarcinoma Moderate 70 wt wt
12 Lung Squamous cell carcinoma Moderate 90 wt wt
13 Lung Adenocarcinoma Moderate 90 wt wt
14 Lung Adenocarcinoma Moderate 90 mut (34G>T) mut
15 Colon Adenocarcinoma Moderate 70 mut (34G>T) mut
16 Lung Adenocarcinoma Moderate 95 mut (34G>T) mut
17 Colon Adenocarcinoma Moderate 95 mut (35G>A) mut
18 Lung Adenocarcinoma Poor 85 mut (34G>T) mut
19 Colon Adenocarcinoma Poor 80 mut (35G>A) mut
20 Lung Adenocarcinoma Poor 70 mut (34G>T) mut
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Sequencing

PCR reactions were composed of 1X MyTaq Reaction Buffer (Bioline Reagents Ltd., London, UK), 0.4 μmol/L primers (Table 1), 0.05 U/μL MyTaq HS DNA Polymerase (Bioline Reagents Ltd), and 10%/v template cDNA. A GeneAmp PCR System 2700 thermocycler (Applied Biosystems, Foster City, CA) was used with the following thermocycling conditions: 2 minutes at 95°C; followed by 45 cycles of 15 seconds at 95°C, 30 seconds at 57°C, and 30 seconds at 72°C; then finally 2 minutes at 72°C. PCR products were analyzed using Agilent 2100 DNA 1000 Series II kits and instrumentation (Agilent Technologies, Santa Clara, CA) to confirm amplification and estimate amplicon concentration. The products were then purified using the QIAquick PCR Purification Kit (Qiagen). Sequencing was performed by Eurofins Genomic (Huntsville, AL) using the reverse-sequencing PCR primer.

PCR for Generation of HIAMD Targets

PCR reactions were composed of 1X MyTaq Reaction Buffer (Bioline Reagents Ltd), 0.4 μmol/L primers (sequences described previously26) (Table 1), 0.05 U/μL MyTaq HS DNA Polymerase (Bioline Reagents Ltd), and 10%/v template cDNA. A GeneAmp PCR System 2700 thermocycler (Applied Biosystems) was used with the following thermocycling conditions: 3 minutes at 95°C; followed by 50 cycles of 15 seconds at 95°C, 15 seconds at 58°C, and 5 seconds at 72°C; then finally 2 minutes at 72°C. The PCR products were analyzed using Agilent 2100 DNA 1000 Series II kits and instrumentation (Agilent Technologies) to confirm amplification. Before HIAMD analysis, the PCR product was denatured at 95°C for 2 minutes and snap-cooled on ice.

HIAMD Instrumentation and Mircowell Chip

Instrumentation was developed in-house and described previously.27 Briefly, the setup consists of a vortex mixer (MS 3 Basic Vortex Mixer; IKA, Wilmington, NC) to hold the chip and provide gentle agitation, and a rotating magnet positioned above the chip to provide additional mixing of the probe-bound magnetic beads. Each chip (4 cm × 4 cm × 1.5 mm) was made of two layers of a plastic substrate [poly(methyl methacrylate)] (Astra Products, Baldwin, NY) and featured a 12-well circular array of 5-mm (diameter) circular wells. A detailed microdevice fabrication method was described previously.22

Bead Preparation

Each set of probe-bound beads was prepared by immobilizing biotinylated probe oligonucleotides to Dynabeads MyOne Streptavidin C1 superparamagnetic beads (InvitroGen, Oslo, Norway). After conjugation and wash steps, the beads were brought up in 1X binding/washing buffer [5 mmol/L Tris-HCl (pH 7.5), 0.5 mmol/L EDTA, 1 mol/L NaCl] in a volume equivalent to the initial volume of stock beads, to maintain a concentration of approximately 7 to 10 × 109 beads/mL.

HIAMD Assay

Each HIAMD reaction took place in a 5-mm (diameter) circular well in a total volume of 20 μL, composed of 10 μL of target sample (either PCR product or synthetic target sequence at a concentration of 1 × 1011 copies/μL, corresponding to approximately 50 ng of input DNA), 9 μL of hybridization buffer (50 mmol/L KCl, 2.5 mmol/L Tris, and 75%/v formamide), and 1 μL of probe-bound beads (equal parts of the two probe-bound beads). (Note that lesser concentrations of target sample in the assay may produce unreliable results.22) The chip was placed on the HIAMD setup, using a rotating magnet speed of 2000 rpm and a vortexing speed of 130 rpm, for a total reaction time of 2 minutes. A digital image of each well was then obtained using a T1i Digital SLR camera with an MP-E 65-mm f/2.8 1−5× macro lens (Canon U.S.A., Inc., Lake Success, NY) and analyzed using a Kapur algorithm in Mathematica software version 8 (Wolfram, Champaign, IL), to derive a quantitative value (saturation) corresponding to the extent of aggregation. The extent of aggregation from each sample was normalized (as a percentage) to the aggregation from the synthetic wild-type sequence. The aggregation measured in a blank sample (the background) was subtracted from this normalized value to yield the final percentage-aggregation value.

Results

KRAS Mutation Analysis in Lung and Colorectal Cancer Cell Lines

To ensure the feasibility of using HIAMD technology for assessing KRAS mutation status in lung and colorectal cancers, we applied the method for the analysis of a panel of lung and colorectal cancer cell lines. For both lung and colorectal cancers, wild-type and mutant cell lines were tested. The percentage-aggregation values measured from KRAS mutant cell lines (SW-620 and H2122) were significantly less than the percentage aggregation from KRAS wild-type cell lines (CaCo2 and H1975, Figure 2). Both mutant cell lines displayed percentage-aggregation values near zero, indicating that the extent of bead aggregation induced by these samples was similar to that of blank (no DNA) samples; this finding provides evidence of the high selectivity of the assay. Importantly, the assay was useful for distinguishing mutations in both the SW-620 and H2122 cell lines, even though the mutations are present at different positions in the gene (c.35 and c.34, respectively).

Figure 2.

Figure 2

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KRAS mutation analysis of lung cancer and colorectal cancer cell lines. Using a PCR-derived target sequence, each cell line was analyzed using the HIAMD assay. The results indicate a significant decrease in the extent of aggregation produced by cell lines bearing a KRAS mutation (SW-620 and H2122) as compared to wild-type cell lines (CaCo2 and H1975). Data are expressed as means ± SD. n = 3 per group.

Resolution of Mutant Alleles in the Background of Wild-Type

In cancer, the mutated cells are surrounded by stroma, which include cancer-associated fibroblasts, endothelium, and immune cells—all of which harbor wild-type KRAS alleles. Therefore, we investigated the utility of HIAMD in detecting a KRAS mutation in a background of wild-type DNA. Synthetic mutant targets were diluted with synthetic wild-type targets to model mixed samples of the following compositions: 75% mutant, 50% mutant, and 25% mutant. These samples were assayed on the same multiwell chip along with 100% mutant, 0% mutant, and blank (no DNA) samples. All samples with mutant content were distinguishable from the wild-type sample, including the sample with only 25% mutant DNA (Figure 3). This detection limit is comparable to that of sequencing, which is reported to be in the range of 15% to 30%.17, 19, 28 In terms of tumor purity, if we consider a heterozygous genotype of the cancer (allele frequency of 0.5), the tumor purity would need to be at least 50% to have at least 25% mutant content.

Figure 3.

Figure 3

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Resolution of mutant (mut) DNA in a background of wild-type (WT) DNA. HIAMD was applied for the analysis of synthetic samples with mixed genotypes. All samples with mutant content (down to 25% mutant) were significantly distinguishable from a wild-type sample. Data are expressed as means ± SD. n = 3 per group.

A threshold value of 36% aggregation was calculated based on the results of the 100% mutant sample, using the mean aggregation value minus three times the SD. Thus, any aggregation value <36% would be associated with a mutant genotype.

KRAS Mutation Analysis of Patients' Samples

Frozen tissue samples from 20 patients, including 10 lung and 10 colorectal surgical resections, were obtained from the Department of Pathology, University of Virginia (Table 2). All tissue samples showed pathologic abnormality, with diagnoses of adenocarcinoma (n = 17) or squamous cell carcinoma (n = 3). The samples were collected and processed using standard pathology workflow. KRAS mutation analysis was performed for each sample in parallel, via sequencing and HIAMD.

Sequencing results indicated that 7 (3 colorectal and 4 lung) of the 20 samples (35%) harbored a KRAS mutation. Of the mutant samples, five were 34G>T and two were 35G>A, representing 71.4% and 28.6%, respectively. Notably, all samples that appeared to have a KRAS mutation also contained wild-type DNA based on the sequencing electropherograms (Figure 4A). This finding was expected for patients' tumor samples, and therefore, we highlight that our sample set, although limited in size, serves as a representative collection of typical clinical samples.

Figure 4.

Figure 4

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KRAS mutation analysis of patient tissue samples. A: Representative sequencing results of three patient samples. (Note: the reverse strand was sequenced). B: HIAMD results. Aggregation of 36% was used as the threshold value to distinguish between wild-type (≥36%) and mutant (<36%) genotypes. Patient numbers are displayed on the x axis. Data are expressed as means ± SD.

HIAMD assays were performed, and percentage-aggregation values of all tumor DNA samples were calculated and plotted (Figure 4B). The x axis was set to cross at the set threshold value (36% aggregation) for ease of data interpretation. Samples with a wild-type KRAS genotype have aggregation values ≥36% and therefore lie above the x axis, whereas samples harboring a KRAS mutation result in aggregation values <36% and therefore fall below the x axis. In all 20 samples, the results of HIAMD analysis were in agreement with sequencing; thus, we report the sensitivity and specificity of our method for the assayed 20 samples to be 100%. Notably, the performance of the HIAMD method was unaffected by the pathologic classification of the tumor (ie, adenocarcinoma versus squamous cell carcinoma) or the position and base substitution associated with the mutation (ie, 34G>T versus 35G>A).

Discussion

Targeted therapies are a growing trend in basic and clinical cancer research, and for good reason—the potential for improved treatment outcomes and cost-savings is tremendous. The effective implementation of a targeted therapeutic regimen requires a practical means of preemptive molecular characterization of the cancer. Here we have shown that HIAMD can be applied for KRAS mutation analysis of primary tumors, which is essential for predicting a patient's sensitivity to epidermal growth factor receptor–mediated therapies,29 as well as population stratification in ongoing therapeutic development.14 Importantly, the analysis is performed in a manner that is both rapid and cost-effective. Although a larger study is warranted for validation, this work establishes the potential of applying the HIAMD technology for routine analysis of other, increasingly important genetic markers.

The initial mutation analysis of cell lines described in this work shows proof-of-concept for KRAS mutation detection with the HIAMD technology in lung and colorectal cancers. However, the value of KRAS mutation analysis requires that the method be validated for use in patients' tumor samples. Unlike samples from cell lines, tumor samples are complex and heterogeneous, and variably contaminated with nontumor content. Thus, the requirements of a mutation-testing method with respect to analytical performance are considerably more demanding and more difficult to achieve without great technical complexity.

Ultimately, we show a 100% correlation between results derived from sequencing and HIAMD analysis for KRAS mutation screening of 20 lung and colorectal tumors from patients. Furthermore, we show that a sample with only 25% KRAS mutant content can be detected in a background of wild-type DNA, which is consistent with the detection limit reported using the more costly and cumbersome sequencing method.19, 28 Thus, we demonstrate that, using HIAMD technology, mutation detection can be performed rapidly and inexpensively without compromising the analytical performance.

Undoubtedly, there are a number of technologies capable of revealing point mutations with higher sensitivity (eg, next-generation sequencing platforms30), which may be preferred for the analysis of very low–purity samples. However, it must be emphasized that, although impressive, high sensitivity alone does not render a technology appropriate for routine clinical screening. Significant consideration must be given to the cost, complexity, and speed of the method. The inherent features of the HIAMD technology make it a natural fit for use in the routine diagnostic setting. Fundamentally, the method is simple. The instrumentation comprises nothing less than a commercial vortex mixer, a rotating magnet obtained from conventional laboratory equipment,27 a reusable plastic microwell chip, a camera, and a laptop computer. The simple instrumentation keeps the method very low in cost (approximately $2500 for all equipment, compared to approximately $100,000 for sequencing instrumentation18), and therefore decreases the financial burden on the patient and the health care provider. In addition, the simple analytical approach translates to simple technical operation, data analysis, and interpretation, therefore eliminating the need for highly skilled personnel. Furthermore, because the simple optical-analysis strategy requires only a camera and a laptop computer, the potential for using a cell phone for detection is high.31 This transition is an obvious next step and will only increase the suitability of this technology for the point of care.

The HIAMD method uses samples processed according to current clinical protocols and is compatible with the clinical laboratory workflow for sample preparation, which includes nucleic acid extraction and PCR amplification. After amplification, HIAMD analysis for the detection of all common KRAS mutations (located in codons 12 and 13) is complete in <10 minutes, which includes approximately 3 minutes for thermal denaturation and snap-cool, approximately 1 minute for pipetting the reagents (sample, buffer, and beads) onto the chip, 2 minutes for the hybridization assay, and approximately 3 minutes for image analysis. Sequencing analysis, on the other hand, requires further sample preparation, including a tedious PCR cleanup step, and has a turnaround time in the order of days.19, 32 The 12-well microchip can allow nine patient samples to be analyzed simultaneously, with the inclusion of three controls on each chip (wild-type and mutant positive controls, as well as a negative/no DNA control). Additionally, the input of DNA required for the HIAMD assay is 50 ng, as compared to sequencing and other commercial KRAS assays, which can require from 100 to 1600 ng of input DNA.32 Thus, speed, low labor requirements, multiplexing capabilities, and low DNA input are all strategic advantages offered by the HIAMD method and contribute to its suitability for clinical implementation.

With the HIAMD method, confirmation with sequencing may be warranted for samples displaying mutant profiles, as has been suggested for other screening methods.33 However, at a cost of approximately $0.75 per test, HIAMD is an economical choice for first screening of mutations and eliminating the cost and lengthy time-to-result of sequencing for the majority of colorectal cancer and lung cancer patients who harbor wild-type KRAS. Alternatively, additional reactions using hybridization probes designed for the positive identification of KRAS mutations could be incorporated onto the chip to increase the reliability of the results.

We show here that HIAMD has true potential for integration into current clinical paradigms, addressing the demand for a practical routine testing methodology. The current direction of clinical oncology research suggests that a technology such as HIAMD will continue to be a highly relevant and valued analytical tool for the facilitation of individualized therapeutic strategies.

Acknowledgments

We thank Agilent Technologies for donating the DNA 1000 kits and the UVa Biorepository and Tissue Research Facility—specifically, Craig Rumpel for arranging the procurement of tumor specimens and Dr. Pat Pramoonjago for helping with RNA extraction.

Footnotes

Supported by NIH NCI Cancer Center support grant P30 CA44579 (University of Virginia Cancer Center).

Disclosures: Agilent Technologies provided the DNA 1000 kits.

References

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