Changes in Chromosome Caps May be A Marker for Tumor Aggression in Neurofibromatosis Type 1
Researchers at the Johns Hopkins Kimmel Cancer Center report that their study of tumor samples from people with the rare genetic syndrome neurofibromatosis type 1 (NF1) has uncovered novel molecular clues about which tumors are most ly to be aggressive in those with NF1. According to the researchers, the clues could advance the search for more customized and relevant treatments that spare patients exposure to treatments unly to work.
“Some NF1 tumors are hard to classify as benign or malignant,” says study leader Fausto Rodriguez, M.D., associate professor of pathology, oncology, and ophthalmology at the Johns Hopkins University School of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. “Having another molecular marker to evaluate will help us make better predictions about the outcomes of treatment.”
A summary of the study was published online in the journal Acta Neuropathologica Communications on Aug. 28.
NF1 is caused by mutations in the NF1 gene. Although rare overall, occurring in about one in 3,000 people, it is among the most common so-called genetic syndromes. About half of all cases are inherited.
People with NF1 are predisposed to develop multiple tumors in the brain and along peripheral nerves throughout the body, just under the skin, and also are predisposed to form rarer tumors of the gastrointestinal tract, adrenal gland and other soft tissues.
The overall number of tumors per patient varies greatly, from several to hundreds over their lifetimes.
While most are slow-growing (low-grade) and benign, some benign peripheral nerve tumors (neurofibromas) develop into so-called malignant peripheral nerve sheath tumors (MPNST), which metastasize frequently, are challenging to treat and are a major cause of death in patients with NF1.
Over time, Rodriguez said he noticed that many of the high-grade, aggressive NF1 tumors he examined were lacking the protein ATRX, a loss associated with a cancer-specific biological process called ALT, short for alternative lengthening of telomeres. Co-investigator Christopher Heaphy, Ph.D.
, ALT expert and assistant professor of pathology and oncology at Johns Hopkins, explains that at the end of each chromosome in a cell’s nucleus, a sequence of six DNA “letters” is repeated up to 2,000 times to form a so-called telomere. “Telomeres are the plastic caps on the ends of shoelaces,” he says.
“They protect chromosomes from fraying or getting jumbled.”
Before a cell divides, it must duplicate its chromosomes, along with their telomeres, but the very tips of the telomeres can’t be replicated. So a few dozen DNA letters are lost each time a cell divides, making telomere length a stand-in for the age of a cell, says Heaphy.
Since cancer cells divide more frequently than most healthy cells, their telomeres shorten more quickly, putting the cell in danger of chromosomal instability, cell death and “genomic catastrophe,” says Heaphy.
To combat this, most cancer cells turn on a special enzyme called telomerase to maintain the length of their telomeres. (Carol Greider, Ph.D.
, the Daniel Nathans Professor and Director of Molecular Biology and Genetics at Johns Hopkins, won the 2009 Nobel Prize in Physiology or Medicine for her discovery of telomerase.)
“A subset of cancers, however, do not activate telomerase but use the ALT mechanism instead,” says Heaphy. ALT uses homologous recombination, a type of exchange of genetic material, to lengthen telomeres, but how exactly this happens remains under investigation.
To assess the importance of ALT in NF1 tumors, Rodriguez, Heaphy and colleagues used fluorescent probes that illuminated the telomeres to examine 426 tumor samples from 256 patients with NF1.
Overall, ALT was detected in 23 NF1-associated gliomas (32.9%). Of the ALT-positive tumors, 14 (60%) were high-grade, or aggressive, tumors, while only 9 of 47 low grade gliomas (19%) were ALT positive.
Among the 99 MPNST samples, 93 developed in patients known to have NF1. ALT was present in 15 of 148 (17%) patients, but ALT was almost entirely absent from the 77 neurofibromas studied and all other rarer tumors (46), except for two cases, which were both malignant.
“The data show that ALT is usually — although not always — associated with more aggressive tumors in patients with NF1,” says Rodriguez. “It’s also more ly to occur in gliomas than MPNSTs.”
Using the same assay, the team also assessed cells from tumors that were ALT-negative. They assessed the overall telomere lengths and compared them with those in non-cancerous cells within the same sample, and then scored as short, normal or long.
In the 183 ALT-negative neurofibromas assessed, all the telomere lengths were considered normal. By contrast, normal telomeres were found in only 57% and 50%, respectively, of the 30 ALT-negative gliomas and 30 ALT-negative MPNSTs tested. While altered telomere lengths were prevalent, abnormal telomeres tended to be too long in gliomas (40%), and too short in MPNSTs (47%).
Rodriguez says those differences could be used clinically to predict tumor aggressiveness, but he cautioned that the differences also could stem from variations in patient age, the fact that standard telomere lengths differ in various cell types, the timing of telomerase activation, or something else. Larger studies are needed, he adds, to rule out those possibly confounding factors.
Finally, the researchers analyzed correlations between telomere alterations and patient survival. Patients with gliomas survived longer if they were ALT-negative even when adjusting for age and grade, two important factors affecting outcome in brain tumors.
For patients with MPNSTs there was no statistically significant difference between those that were ALT-positive or negative.
However, among the whole group of MPNSTs, the researchers found that short telomeres appeared to provide a significant survival advantage, a finding they called “counterintuitive” and that remains unexplained.
“While ALT analysis is not yet a clinical test, surrogate markers such as ATRX loss, may be useful in NF1 patients as a prognostic indicator of more aggressive tumors and worse survival,” says Rodriguez.
In addition to Rodriquez and Heaphy, other researchers included Mindy Graham, Jacqueline Brosnan-Cashman, John Barber, Christine Davis, M. Adelita Vizcaino, Doreen Palsgrove, Caterina Giannini, Me Pekmezci, Sonika Dahiya, Murat Gokden, Michael Noë, Laura Wood, Christine Pratilas, Carol Morris, Allan Belzberg and Jaishri Blakeley.
The research was funded by Pilocytic/Pilomyxoid Research Fund, including Lauren’s First and Goal, and the Stick it to Brain Tumors Annual Women’s Ice Hockey Tournament; the Basic/Translational Science Investigator Award from the North American Neuroendocrine Tumor Society supported by the Neuroendocrine Tumor Research Foundation; a collaborative grant between the Wilmer Eye Institute and the King Khaled Eye Specialist Hospital in Riyadh, Saudi Arabia; Department of Defense grant W81XWH-18-1-0496; and National Institutes of Health grants 2T32CA009110-39A1 and P30 CA006973.
Nanoparticle Slips Suicide Gene into Pediatric Brain Cancer Cells
If you dispatch a suicide gene, you want to make sure that it bypasses healthy cells on its way to harmful cells, such as cancer cells.
What’s more, you want to make sure that the suicide gene is sent via a delivery system that treads lightly—especially if the suicide gene is meant to treat pediatric patients, who have relatively fragile immune systems.
Unfortunately, pediatric patients may have difficulty tolerating the most common gene delivery systems, which are derived from viruses.
To build a targetable and relatively innocuous delivery system, scientists based at the Johns Hopkins University School of Medicine turned to nanotechnology. Basically, they developed a library of poly(beta-amino ester) nanoparticles, or PBAEs. These tiny delivery vehicles consist of biodegradable, cationic polymers, and they self-assemble with nucleic acids.
According to a study (“Nonviral polymeric nanoparticles for gene therapy in pediatric CNS malignancies”) that appeared in the January issue of Nanomedicine: Nanotechnology, Biology and Medicine, the Johns Hopkins researchers used PBAEs to package and deliver the suicide gene herpes simplex virus type I thymidine kinase (HSVtk). Once HSVtk is delivered to a cancer cell, it makes an enzyme that helps restore the cell’s natural tumor suppression function, promoting apoptosis, the cell’s self-destruct mechanism.
To test their PBAEs, the Johns Hopkins researchers used them to transfect cells in mouse xenograft models of cancer, specifically, models that presented medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT). The researchers confirmed that their PBAE-delivered gene therapy made cancer cells more sensitive to ganciclovir, an antiviral drug.
Ganciclovir is commonly used in gene therapies that deliver an altered HSVtk gene to kill advanced melanoma cells and brain tumor cells. But such therapies typically rely on viral vectors.
“In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT and MB,” the article’s authors wrote. “Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.”
The experiments showed that a combination of the suicide gene and ganciclovir delivered by intraperitoneal injection to mice killed more than 65% of the two types of pediatric brain tumor cells. The combination was deliberately transfected with the gene seven days after the nanoparticle therapy was used to deliver the genetic material.
Mice bearing an AT/RT-type tumor lived 20% longer after receiving the treatment—42 days, compared to 35 days for untreated mice. Those with a group 3 medulloblastoma-type tumor implanted in the brain lived 63% longer, surviving 31 days compared to 19 days for untreated mice.
The authors also indicated that PBAE nanoparticles formulated with plasmid DNA for intracellular gene delivery to pediatric brain cancer cells enabled >50% transfection in both cell lines tested. They also found that the nanoparticles preferentially targeted tumor cells over healthy cells.
The nanoparticles “can carry larger genes than what can be carried by a virus, and can carry combinations of genes,” emphasized Jordan Green, PhD, a co-corresponding author of the current study and an investigator at the Johns Hopkins Kimmel Cancer Center Bloomberg–Kimmel Institute for Cancer Immunotherapy. “It’s a platform that doesn’t have limitations on the cargo size being delivered, or limitations related to immunogenicity or toxicity. And, it is easier to manufacture than a virus.”
“It’s an exciting alternate way to be able to deliver gene therapy to a tumor in a selective fashion that targets only tumor cells,” added Eric Jackson, MD, another co-corresponding author and an associate professor of neurosurgery at the Johns Hopkins University School of Medicine. “Our idea now is to find other collaborators who may have a gene therapy that they think would work well to kill these tumors.”
Medulloblastoma and AT/RT are two of the most prevalent and deadly pediatric brain malignancies.
Traditional treatments, including radiation, can harm healthy tissue as well as the tumor, and can produce long-lasting developmental side effects in growing children, making it critical to find new therapies, Jackson noted.
Gene therapy that targets only cancer cells is a promising treatment avenue, but many gene therapy methods use a modified virus to deliver their therapeutic payloads of DNA, a method that may not be safe or suitable for pediatric use.
The mechanism that allows the PBAE nanoparticles to target tumor cells preferentially is still being investigated, but Green thinks “the chemical surface of the particle is ly interacting with proteins that are on the surface of certain types of cancer cells.”
“By making small chemical changes to the polymers that make up the nanoparticles, we can significantly change the cellular uptake into particular kinds of cancer cells, and the subsequent gene delivery to the cytosol, in a cell-specific way,” Green asserted.
“It should be noted that PBAEs are not restricted to delivering genes such as HSVtk,” the article’s authors added. “[These nanoparticles] have been validated as vectors for the delivery of other nucleic acids such as siRNA19 and miRNA24 as well.”
Jackson said that he hopes the nanoparticles can be used to deliver a variety of gene-based treatments—including therapies that alter the expression levels of genes, turn genes on and off altogether, or sensitize cells to other therapies—depending on the specifics of a patient’s tumor. “In some ways,” he remarked, “we’re still in the discovery phase of what genes to target” in medulloblastoma and AT/RT.
Ludwig Cancer Research
Ludwig Johns Hopkins Co-director Bert Vogelstein and Ludwig Stanford Director Irv Weissman have won the 2019 Albany Medical Center Prize in Medicine and Biomedical Research.
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A trust fund created by billionaire shipping tycoon Daniel K. Ludwig ends today with a bang and a gift to research. Six U.S. medical centers will receive $540 million—$90 million each—from the fund to endow cancer studies in perpetuity.
Scientists from the Ludwig Institute for Cancer Research and the Karolinska Institutet report the development of an improved method for analyzing the genes expressed within a single cell. They say their finding will be relevant for everything from basic research to future cancer diagnostics.
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