Because of Kim’s long commitment to treating mycosis fungoides and Sézary syndrome at Stanford, Khavari and his team were able to recruit 91 patients to sequence specific regions of the T cells’ DNA they suspected might be modified in the cancer. From each patient, they collected both cancerous and healthy cells to compare their DNA. This way, they were able to discount any inherent mutations present in the patient before cancer. They identified 170 genes with mutations that could be related to the cancer.
In four of the patients examined, the researchers identified a mutation that replaced a specific amino acid in the tumor necrosis factor receptor 2, a protein embedded in the cell’s membrane that receives signals from outside the cell. The mutation locked the receptor into an always-on state, preventing the cell-survival pathway from shutting down. Previous independent clinical studies found patients with increased TNFR2 protein in their bloodstream had more aggressive forms of the cancer that were more likely to return quickly after treatment. This led Khavari’s team to look at the other patients’ DNA to see if duplications could account for both the elevated levels in the blood and increased signaling to activate the cell-survival pathway. They found that 10 of the patients had multiple copies of the TNFR2 gene.
The researchers confirmed the biological role of TNFR2 by growing cells in the lab with either the amino acid mutation or the duplicate TNFR2 genes and showing the cell-survival pathway to be more active than normal cells.
Although only 5 percent of the cancers had the TNFR2 mutation, the fact that it was the exact same mutation was a “smoking gun,” according to Khavari, implicating the cell-survival mechanism’s role in driving certain cutaneous T-cell lymphomas. While over half of the patients with the disease did not have these gene changes, identifying those who do presents new options for treating them.
Another one of the mutations caused a receptor that normally signals the cell-survival pathway to stop to instead activate it further and encourage cell proliferation. This receptor, CTLA4, has been identified in skin cancers, and an antibody that turns off the receptor has been approved as the drug ipilimumab to treat advanced melanoma. But before administering the drug, or less toxic alternatives, a physician would need to know if the patient had the mutated receptor; otherwise ipilimumab would have the opposite effect, deactivate a healthy protein and make the cancer worse.
University of California-San Francisco melanoma specialist Susana Ortiz-Urda, MD, PhD, who was not involved with the study, called the work groundbreaking and said she was impressed that the researchers were able to gather so many patients to identify the rare mutations. Ortiz-Urda, who co-directs the UCSF Melanoma Center, said she thought the next step was “putting the paper to work in a clinical setting” to see if patients with different mutations would respond to different drug treatments.
Before we had this data, it was trial and error — we were totally blind.
Kim, the Joanne and Peter Haas Jr. Professor in Cutaneous Lymphoma Research who directs Stanford’s Multidisciplinary Cutaneous Lymphoma Program, plans to do just that, using the individual patients’ cancer cell genetic sequences to design combinations of drugs that would hit multiple defective proteins to completely shut down the cell-survival mechanism. Khavari’s lab will be working to incorporate the mutations they identified into the DNA of living mice. This will allow them to study the mutated genes’ effects, and the actions of new drugs on those genes, directly.
“Before we had this data, it was trial and error — we were totally blind,” said Kim. “We’re finally taking the blindfolds off.”
Other Stanford-affiliated authors are postdoctoral scholars Carolyn Lee, MD, PhD, Ashley Zehnder, DVM, PhD, Jason Reuter, PhD, and Mahkam Tavallaee, PhD; research assistant Angela Mah; Michael Snyder, PhD, professor of genetics; Robert Ohgami, MD, PhD, clinical instructor of pathology; Dita Gratzinger, MD, PhD, assistant professor of pathology; and flow cytometrist Randall Armstrong.
This study was funded by the National Institutes of Health (grants R01CA142635, F32CA168091, ASHRTAF and F310CA180408), the Office of Research and Development of the U.S. Department of Veterans Affairs, the Dermatology Foundation, the Haas Family Foundation, and the Drs. Martin and Dorothy Spatz Charitable Foundation.
Information about Stanford’s Department of Dermatology, which also supported the work, is available at http://dermatology.stanford.edu.
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