Digital PCR technology detects brain-tumour-associated mutation in cerebrospinal fluid

Massachusetts General Hospital (MGH) researchers and their colleagues have used digital versions of a standard molecular biology tool to detect a common tumour-associated mutation in the cerebrospinal fluid (CSF) of patients with brain tumours. In their report, the investigators describe using advanced forms of the gene-amplification technology polymerase chain reaction (PCR) to analyse bits of RNA carried in membrane-covered sacs called extracellular vesicles for the presence of a tumour-associated mutation in a gene called IDH1.
‘Reliable detection of tumour-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumours 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 tumours could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme.’
Both normal and tumour 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 tumour-associated mutation in extracellular vesicles from the blood of brain tumour patients, but most current diagnostic technologies that analyse CSF do not capture molecular or genetic information from central nervous system tumours.
In addition, explains Leonora Balaj, PhD, of MGH Neurology, co-lead author of the current report, ‘Tumour-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 tumour-specific changes without the need for biopsy.’
The current study used two forms of digital PCR – BEAMing and Droplet Digital PCR – to analyse extracellular vesicles in the blood and CSF of brain tumour 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 tumours. Two of the three mutation-positive tumours 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 tumours affect the ability to detect mutations. The failure to detect tumour-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumours.
The ability to non-invasively determine the genetic makeup of brain tumours 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 tumour 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 tumour prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumour.
‘Having this sort of molecular diagnostic assay – whether in spinal fluid or blood – would allow us to immediately initiate treatment that is personalised for that patient without the need for surgical biopsy,’ he adds. ‘For some patients, the treatment could shrink a tumour before surgical removal, for others it may control tumour 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.’ Massachusetts General Hospital