Dr. Yaël Mossé is the study co-leader on a research article titled “Differential Inhibitor Sensitivity of Anaplastic Lymphoma Kinase (ALK) Variants Found in Neuroblastoma,” published in Science Translational Medicine in November 2011. Results of her research suggest that either increasing the dose of crizotinib or engineering higher-affinity inhibitors should improve therapy for patients with one of two common ALK mutations found in neuroblastoma. Read on for more details about this research and what it might mean for treatment of children with neuroblastoma who have an abnormality in the ALK gene.
Mossé: It was 2008 when we first found that the inherited form of neuroblastoma is caused by a mutation of the ALK gene. We began identifying the full spectrum of ALK mutations and determining how often they are found in children with neuroblastoma. We found many mutations, but two in particular were found most frequently — the R1275Q and F1174L variants. We focused in on these two mutations, and began examining their response to the ALK inhibitor crizotinib. Crizotinib is an FDA-approved drug used to treat adults with non-small cell lung cancer, a disease that in a subset of patients is caused by translocations of the ALK gene. These new findings are the result of three years of research, and could not have been done without the many more years of investigation that preceded it.
Mossé: An important part of this research was the early observation in our lab that neuroblastomas with varying ALK mutations differed in their response to treatment with crozitinib. Once we saw that the F1174L mutation was far less sensitive to the drug, we predicted that upon further analysis, we’d see differences in how the drug binds to the ALK protein. That was completely wrong. Instead, in collaboration with Dr. Mark Lemmon at the University of Pennsylvania we found that it just takes much higher doses of the drug to see a response. It was a surprise, but great news. That result suggested that crizotinib could potentially be used to treat children with neuroblastoma who have the more resistant ALK mutation in their tumor. This finding is also likely to be relevant for patients with ALK-positive non-small cell lung cancer, and higher doses may delay or prevent the development of resistance.
Mossé: No, this is truly a paradigm shift. This emphasizes the importance of integrating discoveries made in the lab with early clinical studies, which is exciting. As we realized that we were finding new information about neuroblastoma cells that depend on ALK and that could make a significant difference, we were able to move approvals through the appropriate scientific and regulatory bodies to test substantially higher doses of crizotinib in the phase I trial, changing the way we think about clinical trial design. In addition, we have also made it mandatory that we record the blood levels of crizotinib in each child enrolled in the trial, and with the expertise of Drs. Frank Balis and Beth Fox, we will be able to continue to integrate the results from the clinical trial with our laboratory data. We only have one opportunity to define the “right” dose of crizotinib, a critical step to integrating this drug upfront for newly diagnosed patients who we think may benefit from an ALK inhibitor.
Mossé: As we increase the dose of crizotinib given to children in clinical trials, we plan to look at the concentration of the drug in their blood. We’ll bring that information back to the lab and compare the concentration in humans to the concentration that was found to be effective at killing cancer cells in mouse models. By doing this, we’ll be able to establish the recommended dose of crizotinib for pediatric neuroblastoma patients who have this resistant ALK mutation. We’ll also need to find out whether crizotinib can be safely combined with chemo.
Mossé: The assays we used were conventional, but unique to our approach is the teaming up of basic and translational researchers inspired to use their science to impact patient care. We have been working with neuroblastoma cell lines derived from patients with the disease for a long time, and know that most are from patients with very aggressive neuroblastoma that has shown to be quite resistant to treatment. When we poured this ALK inhibitor over these cells and saw what it did, we knew we’d just witnessed something important, and that we needed to know more about it. The team at CHOP could not have done this on our own. Collaborative science was critical to learning what we did about the various ALK mutations and how they respond to crizotinib. In particular, Dr. Mark Lemmon, a Professor of Biochemistry and Biophysics at the University of Pennsylvania, brought to the team his structural knowledge of genes known to cause cancer, an expertise and insight into the biochemical properties of cancer genes that was essential. We’re surrounded by brilliant scientists and clinicians here in Philadelphia and around the world, and it’s imperative that we put our heads together to keep moving closer to cures for pediatric cancer.
These graphs show how studies in mice illustrate how human mutations in the ALK gene affect response to the anti-cancer drug crizotinib. In all four graphs, mice treated with crizotinib (red lines) are compared to mice treated with placebo (blue lines). The top graphs show results on mice with the most common human ALK mutation, R1275Q, which is sensitive to crizotinib. The bottom graphs show mice with the second most common mutation, F1174L, which is more resistant to crizotinib.
In mice with the R1275Q mutation, tumors shrink dramatically (fig. 1) compared to controls, and mice have high survival rates (fig. 2) compared to controls, all of which died. In contrast, mice with the F1174 mutation have a delay in tumor growth but no tumor shrinkage compared to controls (fig. 3) and zero survival (fig 4).
In their research study, Drs. Mossé and Lemmon described the molecular events that account for how the different mutations affect how neuroblastoma tumor cells respond to the drug. The scientists concluded that children having the drug-resistant mutation (F1174L) could benefit from a higher dose of crizotinib. A future pediatric clinical trial will test this approach.
Crizotinib was originally designed as an inhibitor of the MET oncogene, but was subsequently found to inhibit the anaplastic lymphoma kinase (ALK) oncogene. It works by binding within the ATP-binding pocket of these kinases. This competes with protein kinases, that when mutated, cause unregulated reproduction of cells. Crizotinib received accelerated FDA-approved to treat certain non-small cell lung cancers in August 2011. Drugs that target specific ATP-binding pockets first entered clinical trials in the 1990s. Prior to that, it was believed that it would be too difficult to design such a compound. There are hundreds of kinases in the human body, and each has its own ATP-binding pocket. But as scientists learned more about the varying structure of ATP-binding pockets, they realized that it would be possible to inhibit a specific enzyme found in a cancer cell, rather than killing all cells in the body that divide rapidly. The first such drug was Gleevec, which received FDA approval in May 2001 for treatment of patients with chronic myelogenous leukemia.
About 4 to 8 percent of patients with non-small cell lung cancers have a chromosomal rearrangement that generates a fusion gene between EML4 (echinoderm microtubule-associated protein-like 4) and ALK. The resulting kinase activity contributes to the development of cancer. Scientists at the Center for Childhood Cancer Research found in 2008 that the majority of hereditary neuroblastomas are caused by germline mutations in the ALK gene, leading to changes in the blueprint of this gene that alter the genetic message and lead to initiation of neuroblastoma. These mutations result in constitutive phosphorylation — unregulated growth of the cell. It was shown that targeted knockdown of ALK messenger RNA stopped growth in all cell lines where mutant or amplified ALK was present, as well as in two out of six wild-type cell lines for ALK. This can be achieved with an ALK inhibitor, such as crizotinib. The discovery of ALK mutations in February 2008 was translated into the activation of a phase 1/2 trial of Crizotinib within the Children’s Oncology Group in August 2009.
All children with a diagnosis of neuroblastoma should be tested for the ALK mutation, something that CHOP currently offers for families across the country. Having this information provides potentially more treatment options for children who have suffered a relapse, with the ultimate goal of customizing the approach when patients are newly diagnosed with the type of neuroblastoma that has an ALK abnormality.
CHOP’s molecular genetics lab offers several diagnostic tests related to neuroblastoma. The following links provide all information about these tests and how to submit a specimen.
All specimens submitted for testing require a Molecular Genetics Requisition Form and Molecular Genetics Informed Consent. If you have any questions about these diagnostic tests, please contact Catherine Stokes at 215-590-8736 (Office), 215-590-4404 (Lab) or, firstname.lastname@example.org.
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