Massachusetts General Hospital (MGH) researchers and their colleagues have used digital versions of a standard molecular biology tool to detect a common tumor-associated mutation in the cerebrospinal fluid (CSF) of patients with brain tumors. In their report being published in the open-access journal Molecular Therapy – Nucleic Acids, the investigators describe using advanced forms of the gene-amplification technology polymerase chain reaction (PCR) to analyze bits of RNA carried in membrane-covered sacs called extracellular vesicles for the presence of a tumor-associated mutation in a gene called IDH1.
"Reliable detection of tumor-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumors without invasive neurosurgery," says Xandra Breakefield, PhD, of the MGH Molecular Neurogenetics Unit, corresponding author of the paper. "Knowing the IDH1 mutation status of these tumors could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme."
Both normal and tumor cells regularly release extracellular vesicles, which contain segments of RNA, DNA or proteins and can be found in blood, CSF and other body fluids. A 2008 study from the MGH team was able to identify a relatively large tumor-associated mutation in extracellular vesicles from the blood of brain tumor patients, but most current diagnostic technologies that analyze CSF do not capture molecular or genetic information from central nervous system tumors.
In addition, explains Leonora Balaj, PhD, of MGH Neurology, co-lead author of the current report, "Tumor-specific EVs make up only a small percentage of the total number of EVs found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging. These digital PCR techniques allow the amplification of such hard-to-find molecules, dramatically improving the ability to identify tumor-specific changes without the need for biopsy."
The current study used two forms of digital PCR – BEAMing and Droplet Digital PCR – to analyze extracellular vesicles in the blood and CSF of brain tumor patients and healthy controls for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR were able to detect both the presence and abundance of mutant IDH1 in the CSF of 5 of the 8 patients known to have IDH1-mutant tumors. Two of the three mutation-positive tumors that had false negative results were low grade and the third was quite small, suggesting a need for future studies of more samples to determine how the grade and size of the tumors affect the ability to detect mutations. The failure to detect tumor-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumors.
The ability to noninvasively determine the genetic makeup of brain tumors could have a significant effect on patient care, explains study co-author Fred Hochberg, MD, MGH Neurology. "The current approach for patients who may have a brain tumor is first to have a brain scan and then a biopsy to determine whether a growth is malignant. Patients may have a second operation to remove the tumor prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumor.
"Having this sort of molecular diagnostic assay – whether in spinal fluid or blood – would allow us to immediately initiate treatment that is personalized for that patient without the need for surgical biopsy," he adds. "For some patients, the treatment could shrink a tumor before surgical removal, for others it may control tumor growth to the point that surgery is not necessary, which in addition to keeping patients from undergoing an unnecessary procedure, could save costs. We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable."
Breakefield is a professor of Neurology at Harvard Medical School, where Hochberg is an associate professor and Balaj a research fellow, also in Neurology. Walter Chen, MGH Neurology, is also co-lead author of the Molecular Therapy – Nucleic Acid paper. Additional co-authors are Casey Maguire, Li Dan Zhu, and Sarada Sivaraman, MGH Neurology; Linda Liau, Horacio Soto, and Matthew Garrett, University of California, Los Angeles; Michael Samuels and Steven Kotsopoulos, RainDance Technologies; Lori LoGuidice and Johan Skog, Exosome Diagnostics; Clark Chen and Eric Wong, Beth Israel Deaconess Medical Center; and Bob Carter, University of California, San Diego.
The MGH has applied for a patent on the use of BEAMing PCR to analyze RNA from extracellular vesicles. The technology used to isolate tumor-associated molecules from extracellular vesicles, originally developed at the MGH, has been licensed to Exosome Diagnostics. RainDance Technologies markets the Droplet Digital PCR used in this study. Support for the study includes National Institutes of Health grants CA069246, CA141226, CA156009 and CA141150 and grants from the Brain Tumor Funders' Collaborative and the American Brain Tumor Association.
Massachusetts General Hospital (http://www.massgeneral.org), founded in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $775 million and major research centers in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, reproductive biology, systems biology, transplantation biology and photomedicine.
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