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Researchers Use Fluidigm Nanofluidic Digital PCR Array to Quantify Mutations in Lung Cancer Gene

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By Ben Butkus

Researchers from Case Western Reserve University and Fluidigm have published a paper demonstrating how Fluidigm's nanofluidic digital PCR platform can detect and quantify EGFR mutations at the single-molecule level from lung tumors, including formalin-fixed, paraffin-embedded samples.

The method may help provide deeper insight into the specific roles of clinically relevant mutations during different stages of lung tumor progression, and may be useful in predicting the clinical response to EGFR-targeted inhibitors, according to the study.

In addition, the method may be able to supplement current laboratory techniques for oncogene detection and analysis, such as direct DNA sequencing, especially in samples with extremely low levels of genetic material, the researchers said.

The study, published online this month in Clinical Chemistry, sprung from a collaboration between Fluidigm and the laboratory of Patrick Ma, an assistant professor of hematology/oncology at the CWRU School of Medicine and Case Comprehensive Cancer Center.

In an e-mail to PCR Insider this week, Ma said that Fluidigm's digital array technology using digital PCR was a good fit for his group's research on low-abundance mutated alleles in lung tumors, and that it was "a novel platform to quantify both the EGFR relative gene copy number and oncogenic mutated alleles on a single platform assay."

As explained in the paper, targeted therapies such as small-molecule inhibitors of EGFR (epidermal growth factor receptor) have in recent years gained ground on traditional chemotherapy. Small-molecular tyrosine kinase inhibitors such as gefitinib (Iressa) and erlotinib (Tarceva), which target EGFR, have been approved for treating advanced non-small cell lung cancer, which accounts for the majority of lung cancer cases.

However, mutations in EGFR, predominantly in exons 19 and 21, have been identified as mutation hotspots in advanced NSCLC and as having prognostic value. Studies using fluorescence in situ hybridization have shown that detecting an increase in EGFR gene copy number is a strong predictor of survival benefit in patients with NSCLC being treated with EGFR inhibitors.

FISH assays remain the "gold standard" for detecting EGFR copy number, but they are labor-intensive and introduce potential sample bias in determining the mean number of gene copies within a tumor, according to the researchers.

Many other methods have also been studied for detecting EGFR copy number, including DNA amplification followed by direct sequencing, genotyping based on MALDI-TOF mass spectrometry, and the Scorpion amplification refractory mutation system, or ARMS, from Qiagen subsidiary DxS.

Of these, the most commonly used method currently as applied to clinical samples is direct DNA sequencing, Ma said in his e-mail. The test is available under clinical laboratory regulations, but is expensive and detects tumor mutations only within an abundance range of 10 percent to 25 percent, according to the literature.

Meantime, PCR has recently become more popular for detecting EGFR mutations, but still has its limitations, especially when considering samples that are extremely small, compromised, or contain a miniscule amount of genetic material.

Nanofluidic digital PCR array technology, such as that developed by Fluidigm, addresses many of these issues, according to the researchers.

"It can supplement current direct DNA sequencing especially in samples where insufficient DNA materials from minuscule tumor biopsy samples make it impossible to perform direct DNA sequencing," Ma said.

In addition, he added, "the genomic DNA material requirement in the nanofluidic digital PCR platform is much less demanding than the current standard DNA sequencing by capillary gel electrophoresis, which is a great advantage."

In their study, the researchers examined 16 cell lines and 20 samples of genomic DNA from resected tumors in an attempt to detect EGFR alleles and quantify the relative copy number of the EGFR gene in lung cancer.

To detect EGFR alterations, they used Fluidigm's BioMark real-time PCR system and Digital Array chips combined with Scorpion ARMS PCR assays. Whereas conventional digital PCR uses sequential limiting dilutions of target DNA followed by PCR, the digital array performs that same function by partitioning each sample into hundreds of individual PCR reactions in a microfluidic chip.

To quantify the relative EGFR copy number, they used the BioMark system and Digital Array chips with a DxS EGFR29 mutation test kit and a Life Technologies TaqMan assay for the single-copy RPP30 gene, then calculated the ratio of EGFR to RPP30 copies.

Using this method, the researchers were able to detect and quantify rare Iressa/Tarceva-sensitizing EGFR mutations in abundances ranging from 0.02 percent to 9.26 percent that were present in formalin-fixed, paraffin-embedded samples of early-stage lung tumors without using whole-genome amplification methods, which can often introduce bias, according to the paper.

"The Fluidigm nanofluidic digital PCR array platform substantially improves mutation detection assay sensitivity," Ma said. "In addition, it enables investigators to quantify the mutated alleles at a single-molecule level present in the cancer genome, thereby providing great insight into tumor molecular heterogeneity."

Further, the ability of Fluidigm's technology to work on FFPE samples is crucial because FFPE is routinely used to process and store tumor samples in clinical pathology. "Methodologies that are suited to use FFPE samples would [eventually] be more amenable for more universal clinical applicability and translation," Ma said.

In addition, the Fluidigm platform should also be well-suited to oncogenic mutation detection and gene copy number quantification in other scarce tumor specimens "due to its very high assay sensitivity," Ma added. "Examples of potential applications include testing circulating tumor cells, cytology specimens from fine-needle aspirates (instead of core biopsies), or even cerebrospinal fluid tumor cells."

Besides demonstrating the utility of digital nanofluidic PCR in the clinical laboratory setting, the researchers believe they have also added to a growing body of evidence that EGFR mutation analysis may help guide treatment of lung cancer at even earlier stages than previously thought.

"Although … gefitinib and erlotinib were initially approved for treating advanced metastatic NSCLC … much of the current emphasis in clinical investigations is focused on [their] potential use in the first-line setting in selected patients enriched with sensitizing EGFR mutations and as adjuvant therapy in NSCLC patients with tumors resected at an earlier stage," the researchers wrote.

"Knowledge of EGFR-activating mutations in early-stage lung tumors that are sensitive to EGFR TKIs may eventually also allow a paradigm change in the use of these kinase inhibitors in lung cancer treatment of curative intent," they added.

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