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Einstein Researcher Receives Grant to Study Exhaled microRNA as Cancer Biomarker

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NEW YORK (GenomeWeb) – While efforts to use microRNAs as cancer biomarkers have largely focused on analyzing samples of the small, non-coding RNAs from blood or tumor tissue, an Albert Einstein College of Medicine researcher sees potential for miRNAs carried in exhaled breath.

With a two-year grant from the National Cancer Institute, Einstein's Simon Spivack is setting out to validate the results of a pilot study showing that miRNAs could not only be obtained from exhaled breath condensate (EBC), but can indicate when an individual has lung cancer before symptoms arise.

Currently, many lung cancer patients do not seek treatment until their disease has progressed into later stages, making a cure less likely, he told GenomeWeb. As a result, there is a growing focus among those in the medical community on catching diseases early. When it comes to lung cancer, that traditionally means low-dose CT screening for people who smoke and have other risk factors.

Though effective, CT screens are relatively expensive, and oftentimes result in over-diagnosis as they frequently reveal numerous non-cancerous lesions in addition to pre-cancerous ones.

In looking for an alternative for lung cancer risk-assessment, Spivack and his colleagues turned to miRNAs, analyzing samples of non-small cell lung cancer (NSCLC) tumors and adjacent lung tissue for differential miRNA expression.

The researchers took the top 10 hits from this effort and combined them with four other miRNAs that have been associated with NSCLC by other groups to create a 14-miRNA panel, which was used in a pilot study examining the EBC of 95 individuals — half with NSCLC and half who were healthy.

According to Spivack, EBC is obtained by having an individual breath through a mouthpiece connected to a cold collection tube. The condensation that forms on the walls of the tube is then collected and analyzed.

In their pilot study, Spivack and his team used a RNA-specific RT-PCR approach that he first developed in his lab over a decade ago for messenger RNA and later adapted for miRNAs specifically.

The method generally involves using an RT primer that targets the polyA tail of mRNA and adds a unique tag sequence that does not exist in the human genome and is not incorporated by genomic DNA. When PCR is performed using a transcript-specific forward primer and a reverse primer identical to the unique tag sequence, only cDNA made with the RT primer is compatible with the reverse PCR primer, eliminating any confounding signals from contaminating genomic DNA.

After winnowing down their panel of 14 miRNAs to four, the scientists were able to distinguish cancer patients from normal individuals based on EBC with 76 percent accuracy.

The miRNA content of the EBC samples was analyzed using an RNA-specific qPCR technique developed in Spivack's lab that doesn't require DNase treatment. By winnowing down the miRNAs used in the panel from 14 to four, the investigators were able to distinguish NSCLC patients from normal individuals with 76 percent accuracy.

Accuracy improved to 84 percent when simple risk-assessment factors, such as smokers versus non-smokers, were added to the assessment.

Encouraged by these results and with the NCI funding, Spivack's group now aims to test the approach in a greater number of people and under more rigorously designed study conditions.

Specifically, the investigators will work to expand the number of potential biomarker miRNAs by analyzing additional lung tissue samples, identifying miRNA signatures not only from tumors and healthy tissue, but also premalignant lesions.

An expanded miRNA panel will then be tested in a case-controlled study of approximately 300 individuals — half with NSCLC and half without — with the top scoring exhaled miRNAs then being tested in a nested case-control study involving about 200 putative high-risk individuals participating in a prospective lung cancer CT screening study.

If the NCI-funded project yields positive results, the next step would be to move into a prospective trial "where we take large numbers of people, measure their breath once or multiple times over [a period of] time, and see who [develops] lung cancer," Spivack explained.

Spivack's grant began on Dec. 8 and runs until Nov. 30, 2016. It is worth $217,935 in its first year.

EBC attention

Amid a growing interest EBC as a source for lung disease biomarkers, Spivack is not alone in his focus on miRNAs.

In late 2013, an Italian team reported that two miRNAs — miR-21 and miR-486 — could be obtained from both plasma and EBC of NSCLC patients and controls, and that their expression was significantly altered between the two groups.

And earlier this year, a multi-institute team reported that expression of miR-570-3p was found to be significantly lower in EBC of asthmatics compared with healthy individuals, and that the miRNA's expression was inversely correlated to asthmatics' lung function.

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