Cancer researchers yearning for a robust universal biomarker of malignancy and tumor grade have long found telomerase a tempting candidate. This enzyme maintains the telomeres at the tips of chromosomes in early development and stem cells but is then usually inactivated, allowing telomeres to erode and limiting cell division. Most cancer cells, however, reactivate telomerase in their quest to become immortal.
Current methods to measure telomerase levels for clinical diagnosis and prognosis through DNA, RNA, or protein analysis have technical difficulties, including lack of reliable antibodies and inability to use in paraffin-embedded tissues, said Uri Tabori, M.D., from the Hospital for Sick Children in Toronto, Canada. In the past year, he and others took a different approach, looking at the promoter for the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) instead of directly at the gene.
TERT Promoter Methylation Status in Pediatric Brain Tumors
In that study (Lancet Oncol. 2013;14:534–42), Tabori focused on methylation. Although methylation typically silences genes, it can promote cancer, possibly by silencing tumor-suppressor genes. Also, methylation status can be quickly, easily, and accurately tested in clinical samples. In an initial sample of 280 malignant and healthy pediatric brain samples, TERT promoter hypermethylation characterized 72% of the malignant samples but only one healthy sample. In further analyses of different pediatric brain tumors, methylation predicted survival and progression-free survival, increased in tandem with telomerase expression, and distinguished low-grade from high-grade tumors, as well as which low-grade tumors progressed to high-grade lesions.
That finding was exciting because pediatric brain tumors can be difficult to grade, but the distinction has prognostic and therapeutic relevance, explained pediatric oncologist Scott Diede, M.D., Ph.D., of the Fred Hutchinson Cancer Research Center in Seattle, who wrote a commentary on the study (Lancet Oncol. 2013;14:447–8).
“Most low-grade tumors don’t progress, so we wish we could figure out up front which rare tumor we need to treat aggressively instead of waiting until they’ve become more malignant,” he said.
Astoundingly Recurrent TERT Promoter Mutations
Tabori’s methylation paper was already accepted when two studies of melanoma independently finding highly recurrent DNA mutation in the TERT promoter appeared (Science 2013;339:957–9 and [in same issue] 959–61). About half of melanomas have driving mutations in an oncogene called BRAF, which the inhibitor vemurafenib now targets.
“We wondered if mutations in noncoding regions might explain some additional portion of melanomas,” said Levi Garraway, M.D., Ph.D., a cancer geneticist at the Broad Institute of Harvard and MIT. An initial quick sweep of 19 whole-genome sequences of melanoma identified two nearby single-base-pair mutations in 83% of tumors of the TERT promoter. In a larger sample, 50 (71%) of 70 melanoma tumors harbored one or the other mutation.
“We were quite skeptical of this prevalence at first because they could be just an artifact of sequencing,” recalled Franklin Huang, M.D., Ph.D., first author of the paper.
But meticulous follow-up studies confirmed the finding in not only melanoma but also liver and bladder cancers. Moreover, the concurrent Science paper found a similarly high prevalence of the same two mutually exclusive TERT promoter mutations in melanoma.
“We all expected that sooner or later we would find regulatory mutations that were as important as protein-coding mutations,” reflected Garraway, “but we never imagined they would be more prevalent than gene mutations.”
A Theme Emerges: Specificity and Age
“That was a monumental discovery,” said Patrick Killela, a Ph.D. candidate in the lab of Hai Yan, M.D., Ph.D., at Duke University Medical Center and first author of another article on TERT promoter mutations (Proc. Natl. Acad. Sci. USA 2013;110:6021–6). In this study, Duke researchers and their collaborators investigated the TERT promoter mutations in more than 1,200 tumors of 60 types of cancer. Conducting such a large study quickly was possible because the two “hotspot” point mutations are just 22 base pairs apart, so they could be tested by using PCR on one fragment.
“The numbers just jumped off the page,” Killela said.
One or the other TERT promoter mutation occurred in 44% of hepatocellular carcinomas, 66% of bladder cancers, 83% of glioblastomas, and 20% of medulloblastomas—always the most prevalent mutation identified to date. The mutations mostly characterized cancers arising from tissues that self-renew slowly. Previously, researchers had identified several mechanisms such cells use to revert to a more stemlike, proliferative state. Now, TERT promoter aberrations were joining that collection.
Mutation specificity in tumor subtypes also was readily apparent. In gliomas, testing for TERT promoter mutations along with another hotspot mutation, IDH1 or IDH2, could distinguish the most common types of brain tumors: Primary glioblastomas had mutations in TERT only (83%); astrocytomas in IDH only (75%), and 78% of oligodendrogliomas had mutations in both TERT and IDH.
Age of diagnosis also mattered. Whereas 83% of 78 adult primary glioblastoma tumors harbored TERT mutations, only 11% of 19 pediatric glioblastomas did. Likewise in medulloblastoma, typically a pediatric cancer, two of the four subtypes (WNT and SHH) had recurrent mutations, but they were concentrated largely in older patients.
“We were intrigued by their medulloblastoma results,” said pediatric oncologist Marc Remke, M.D., at the Hospital for Sick Children. In a later analysis of 446 medulloblastoma tumors (Acta Neuropathol. 2013;126:917–29), this group also found the mutations concentrated in the WNT (11%) and SHH (38%) subgroups, with a striking age-specific pattern particularly in SHH: Less than 9% of infant, 22% of childhood, and 83% of adult SHH harbored mutations.
Scott Diede, M.D., Ph.D.
“Having a new way to further subdivide the SHH group by TERT promoter mutation opens up a new avenue of biology to explore,” said coauthor Vijay Ramaswarmi, M.D., “and an opportunity for preclinical modeling and targeted therapy.”
Mutations Versus Methylation
What, then, is the relationship between the TERT promoter mutations and the hypermethylation Tabori reported? Are they redundant or alternatives?
“Without knowing about the sequence in the hypermethylation study, we could imagine that DNA methylation was a surrogate for a promoter mutation that they just didn’t know about,” Diede said.
Two studies have now looked at this relationship. The first, led by Koichi Ichimura, M.D., Ph.D., at the National Cancer Center Research Institute in Tokyo, Japan, found that methylation and mutations independently occur in adult gliomas (some tumors have both methylation and mutation) but that only mutations increased TERT expression (Acta Neuropathol. 2013;126:939–41). The second, from Newcastle University in Newcastle-upon-Tyne, UK, analyzed medulloblastoma and observed that mutations and hypermethylation were mutually exclusive and that both increased TERT expression (Acta Neuropathol. 2014;127:307–9). Hypermethylation occurred in various degrees in all subgroups, whereas only noninfant SHH featured mutations.
“I’m not surprised by these discrepancies,” Diede said, “because adult glioma is a totally different brain tumor from medulloblastoma and the pediatric cancers that Tabori’s group had studied.”
Agreeing in an e-mail, Ichimura added, “These studies collectively consolidate the significance of TERT alterations in diverse types of brain tumors, and I expect to see something similar in other types of human cancers too. Our task now is to translate these findings in the clinic to find a way to utilize TERT alterations as a biomarker or therapeutic target.”
But what about that infant SHH, with neither TERT promoter mutation nor methylation, as well as the scarcity of promoter mutations in pediatric gliomas? Perhaps, several researchers speculated, young brain cells already still have active telomerase and do not need any of the aberrations apparently so common among many other brain tumors.
What Else Is Out There?
Just the past year’s cascade of studies, one reacting to another, found an unexpected prevalence of two possibly mutually exclusive TERT promoter aberrations—hypermethylation and point mutations—in multiple cancers. Collectively, these studies validate efforts to move beyond the gene-centric approach to disease research that focuses on protein-coding genes and to embrace noncoding regulatory elements that can initiate and promote cancer.
“We completely missed the TERT promoter in all these years of whole-exome sequencing,” Killela said. “It’s the first promoter we identified, yet it has such significance. How many others are out there?”