Researchers at Uppsala University have developed a new method to identify conserved noncoding mutations in the genome that may play a driving role in medulloblastoma brain tumors in children. The study also linked identified noncoding constraint mutations (NCCMs) with changes to how cancer cells respond to one type of cancer therapy. The researchers suggest that their findings could lead to better diagnostics and more individualized treatment for children with brain tumors. Reporting their study in PNAS, in a paper titled “Using evolutionary constraint to define novel candidate driver genes in medulloblastoma,” co-first author Ananya Roy, PhD, and colleagues concluded that the findings “… may lead to better stratification of medulloblastoma by age and subgroup and may improve targeted treatment strategies.”
The two most common brain tumors in children are pilocytic astrocytoma (PA) and medulloblastoma (MB), the authors further explained. While PA is relatively benign and has few mutations, medulloblastoma is malignant. Medulloblastoma usually develops in the cerebellum, and while modern treatment has improved prognosis such that over 70% of individuals with this type of cancer live for more than five years, not all patients can be cured. Aggressive cancer treatment also causes severe side effects, such as balance problems and impaired learning abilities in cancer survivors.
Numerous studies have explored the less than two percent of human DNA which gives rise to proteins, and much less is known about this remaining 98% of the genome. There could be thousands of mutations, and it is difficult to separate the ones driving the cancer from those without importance. “Identification of cancer driver genes is important for understanding the biology of cancer and to define treatment targets,” the authors wrote. “Cancer driver gene discovery has focused chiefly on coding mutations, with less attention paid to mutations in the noncoding parts, which make up >98% of the genome.”
To gain a comprehensive view of what mutations are important in medulloblastoma, the researchers used a method that they recently developed as part of an international consortium. The method specifically looks at conserved positions in the genome, and builds on the assumption that DNA-sequences that has remained the same over millions of years of evolution are likely to have important functions.
“A systematic way to identify which positions in the genome are important for function can be the study of evolutionary constraint,” the authors commented. “To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation.” To this end, they used whole-genome sequencing data from the International Cancer Genome Consortium, and combined it with phyloP evolutionary constraint scores, inferred from genome studies in 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs) that are likely to be regulatory in nature. “We here apply phyloP scores to whole-genome data of MB and PA to identify genes with increased noncoding constraint mutational burden and potential function in cancer.”
Roy further noted, “In the new study, mutations in patients with medulloblastoma were compared with information about how well an individual position in the genome has been kept throughout evolution. Mutations in areas that hardly changed at all can be assumed to be most important.”
Out of the 200,000 mutations found in 145 patients with medulloblastoma, a total of 114 mutations were found in conserved positions in the genome. Many of these mutations occurred in genes that were previously not reported to be mutated in this cancer, even though some of the genes had altered protein levels in medulloblastoma. The newly identified mutations are located in DNA that contains instructions about where, when and how much of proteins should be made. The results may therefore explain earlier observations of different protein levels in medulloblastoma.
“We focused on mutations at the best-preserved positions, as these are likely the most critical ones for gene regulation,” said study co-lead Karin Forsberg-Nilsson, PhD. “This way we can sort out the most important mutations, which would otherwise not be possible, and then test their functionality.”
The researchers identified different mutations in different age groups—“Most NCCMs associated with adult-onset were found in genes normally expressed in the brain, many with relatively high expression in cerebellum”—and different subgroups of medulloblastoma, and found that the mutations changed gene expression in medulloblastoma cells in culture. “This shows that our method works, and can provide a clearer picture of how these mutations regulate protein levels in cancer cells”, said study co-lead Kerstin Lindblad-Toh, PhD. The investigators also compared medulloblastoma with pilocytic astrocytoma, and found very different NCCM frequencies between the two, which they pointed out is ”in agreement with the fact that malignant cancers tend to have more mutations.”
The researchers also showed that mutations identified using the new method modified the cancer cells’ resistance to the anticancer drug dasatinib. This new way of analyzing mutations could therefore point to more personalized, precision medicine, based on an assessment of the patient’s specific mutation pattern and selection of treatment that will target the mutated gene.
For this approach to work, however, there must be an already existing drug—even one currently used against a different indication—that might be suitable for use in even just a few cancer patients with the appropriate mutation. The new study could potentially also expand the number of treatable mutations, which could ultimately result in better individualized therapies for patients.
“To be able to use cancer mutation analysis for precision medicine, a lot of genetic information about each patient is needed. Since all children with brain tumors in Sweden are now offered whole genome sequencing analysis, i.e., the entire tumor genome is mapped, there are good possibilities to develop the analysis further to benefit patients,” said Forsberg-Nilsson.
In their study the authors stated, “In conclusion, using phyloP constraint scores from 240 mammals, we have shown that genes with NCCMs are associated with different ages of onset and MB subgroups. Several novel candidate driver genes have plausible roles in MB development, and NCCMs could therefore be of diagnostic and/or of therapeutic potential.”
