By Doug Macron
Although diagnostics based on microRNAs obtained from tissue samples have begun to gain traction in the marketplace, many see even greater value in the possibility that the small, non-coding RNAs can be obtained from biofluids for such tests.
To help advance work in this area, the National Institutes of Health recently awarded nearly $500,000 in grant funding to three researchers exploring the use of blood-based miRNAs as cancer biomarkers.
The first grant was issued to Indiana University's Harikrishna Nakshatri to support his investigation into miRNA changes in the serum of disease-free breast cancer patients.
While the source of miRNAs in circulation is unknown, some are thought to be derived from secreted exosomes or released by dying cancer cells, he wrote in his grant's abstract. “Intriguingly, the levels of several microRNAs are lower in the serum of cancer patients compared to healthy [ones], suggesting that cancer is either causing destruction of the adjoining normal tissues from which these microRNAs are normally secreted or it is influencing the expression and secretion of these microRNAs from distant organs.”
Data generated by Nakshatri and colleagues suggests the latter, he noted. In recent studies, serum from breast cancer patients who are clinically disease-free showed elevated levels of two miRNAs, miR-451 and miR-101, but lower levels of four others, miR-370, miR-574-3p, miR-342-3p, and miR-197.
At the same time, serum from these patients contained elevated levels of the RNA polymerase III-transcribed small RNAs U6 and 5S, the abstract states.
In light of these and other findings, Nakshatri hypothesizes that a “chronic inflammation-like condition in cancer patients leads to permanent changes in microRNA/small RNA expression in distant organs, particularly in organs with regenerative capacity such as the liver.
“Cancer or host-response to cancer causes elevated levels of circulating cytokines ... which may mediate the long-term effects of cancer on microRNA expression in distant organs.”
As a result, distinct miRNA and U6 RNA levels persist in the serum of patients who have had cancer but are now disease free, the abstract states.
To test this, he plans to examine whether genes corresponding to miRNAs and small RNAs that are differentially expressed in the serum of animal models show altered expression, histone modifications, and RNA polymerase occupancy in liver and lungs. Serum miRNA profiles and cancer-associated changes in miRNA, U6, and 5s expression will also be determined.
“Serum from breast cancer patients, prior to starting neoadjuvant therapy, immediately after completing therapy but before surgery, and after surgery will be examined for U6, 5S RNA, and select microRNAs,” the abstract notes. “If cancer is the primary source of the above microRNA/small RNAs in the serum, their levels should drop after neoadjuvant therapy and/or surgery. If specific microRNA is host-derived and changes in its expression in distant organs is permanent, therapy should not influence its expression.”
Overall, this work is expected to help determine “cancer-induced collateral damage” to organs and its overall impact on cancer patients, Nakshatri wrote.
The project began on March 9 and runs until the end of February 2013. It is worth $200,970 in its first year.
The second grant was awarded to Columbia University's Regina Santella, who is investigating blood-based miRNAs as an early biomarker of breast cancer.
Studies from various groups have shown abnormal expression levels of both mature precursor miRNA sequences compared with the corresponding normal tissues, she wrote in her grant's abstract. Meanwhile, investigators have demonstrated that stable miRNAs can be found in the blood, all of which suggests that miRNAs in plasma might be useful for early cancer diagnosis.
The NIH grant will support a pilot study to identify candidate miRNA biomarkers over-expressed in the plasma of breast cancer patients, and to help establish laboratory assays for their quantitation.
Specifically, Santella and colleagues will compare whole-genome miRNA expression profiles in 20 plasma samples from breast cancer cases and 20 controls, according to the grant's abstract. The top five to ten miRNAs over-expressed will then be assayed using an RT-qPCR assay to confirm the array data, after which paraffin-embedded breast tumor sections, along with paired adjacent tissues, will be analyzed to determine if the miRNA candidates are also over-expressed in tumor compared with adjacent tissues.
RT-qPCR will be used to confirm plasma differences using an independent set of 100 cases and matched controls.
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“This pilot study will provide the resources to select breast cancer-specific [miRNAs], establish the laboratory assays for quantitation of [miRNAs] in plasma, and demonstrate that we can detect breast cancer at the time of diagnosis,” the abstract adds.
Santella's grant runs from April 1 until March 31, 2013, and is worth $80,125 this year.
University of Texas MD Anderson Cancer Center researcher Wei Zhang received the final grant, which will help fund his efforts to identify blood-based miRNAs associated with colorectal cancer.
“Accumulating evidence has indicated that aberrant expression of miRNAs is associated with cancer development and progression,” he wrote in the grant's abstract. “A successful blood test would be a major help in deciding on neoadjuvant or adjuvant chemotherapy, and would also be used to follow patients for recurrence after curative resection and after chemotherapy for response.”
In a previous pilot study, qRT-PCR-based miRNA assays were used to compare expression of five specific miRNAs in a cohort of 85 plasma samples from healthy donors, colorectal cancer patients with localized disease, and colorectal cancer patients with distant metastatic disease, according to the abstract. “Preliminary data showed that all of these miRNAs can be detected in the plasma. Further, the levels of miR-141, a member of the miR-200 family, were significantly elevated in plasma samples from patients with distant metastatic colorectal cancer.”
Following up on these findings, Zhang aims to use the NIH funding to “identify and put into routine usage a set of robust plasma miRNA markers” for early detection of the cancer, monitoring of early metastasis, and evaluating prognosis.
His grant runs from April 1 until March 31, 2013. It is worth $206,190 in its first year.
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