Researchers have tracked brain tumors by searching for evidence of mutations in cerebrospinal fluid. (Source: Harvard Medical School)

Borrowing a tool from molecular biology, HMS researchers at Massachusetts General Hospital have detected a tumor-associated genetic mutation in the cerebrospinal fluid (CSF) of a small number of patients with brain tumors.  In a paper published in the open-access journal Molecular Therapy – Nucleic Acids, the investigators described using digital versions of the gene-amplification technology polymerase chain reaction (PCR) to analyze bits of RNA carried in membrane-covered sacs. They found a common tumor-associated mutation in a gene called IDH1, a biomarker whose presence could potentially influence patient care.

“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, HMS professor of neurology at Mass General and 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 membrane-covered sacs called extracellular vesicles. Found in blood, CSF and other body fluids, they contain segments of RNA, DNA or proteins. A 2008 study from the Mass General team identified 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, “Tumor-specific extracellular vesicles make up only a small percentage of the total number of extracellular vesicles found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging,” explains Leonora Balaj, an HMS research fellow in neurology and co-lead author of the paper. “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 in the CSF of brain tumor patients and healthy controls. The scientists were searching for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR detected the presence and abundance of mutant IDH1 in the CSF of five of the eight 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 tumor 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 impact on patient care. “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,” says Fred Hochberg, HMS associate professor of neurology and a study co-author. “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 kind of molecular diagnostic assay—whether in spinal fluid or blood—would allow clinicians to immediately initiate treatment that is personalized for a particular patient without the need for surgical biopsy, Hochberg says. 

“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,” he says. “We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable.”

Mass General has applied for a patent on the use of BEAMing PCR to analyze RNA from extracellular vesicles. Support for the study includes National Institutes of Health grants and grants from the Brain Tumor Funders’ Collaborative  and the American Brain Tumor Association.