February 2008: Volume 34, Number 2
Targeted Therapeutics in Non-Small Cell Lung Cancer
How Molecular Assays Guide Their Use
By Deborah Levenson
Advanced non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide, is usually diagnosed at a metastatic or advanced stage, when median survival is just eight to 10 months. Therapeutic agents called tyrosine kinase inhibitors (TKIs), small molecules that target the epidermal growth factor receptor (EGFR) gene, can extend survival for certain NSCLC patients. These drugs may be most useful for those NSCLC patients with mutations in particular areas of the gene that encodes EGFR. Present in about 10% of NSCLC cases in North American and Western European patients, these mutations occur at a much higher rates—up to 50%—in Asian countries and in patients of Asian ancestry. The mutations are also more common in women and nonsmokers who have adenocarcinomas with bronchioalveolar features.
Now, updated guidelines from the National Comprehensive Care Network (NCCN), an alliance of 21 leading cancer institutions, are the first to specifically recommend that oncologists consider lab tests to see if one TKI, erlotinib, will be effective in patients with no history of smoking and either a known active EGFR mutation or extra copies of the gene. Erlotinib is marketed as Tarceva in the U.S. by OSI Pharmaceuticals (Melville, N.Y.) through a partnership with Genentech (San Francisco, Calif.). Tests for mutations of the K-ras oncogene, which are more common in patients with a history of smoking, may be useful in determining which patients are unlikely to respond to TKIs.
The tests determine appropriateness of a treatment that offers varying survival benefit, from just a few months to a year or more. And the assays are costly. The EGFR mutation and K-ras assays generally cost between $700 and $1,000 each, while another assay that detects extra copies of the EGFR gene is a bit less, at $400 to $700. But they are worth considering, maintained David S. Ettinger, Chairman of the NCCN committee that wrote the guidelines, and the Alex Grass Professor of Oncology at the Sidney Kimmel Comprehensive Care Center at Johns Hopkins Medical Center in Baltimore, Md. “If you look at the big picture, incremental gains in survival add up. We’re talking about advanced disease here.” He’s hopeful that current trials of approved and newly developed TKIs may yield ther-apies for early stage NSCLC. “We first learn about new agents in advanced disease. As research progresses and we refine the guidelines, we will become more specific in our recommendations,” Ettinger noted. He receives grants support from Genentech and is a member of both its speaker’s bureau and advisory board.
Guidelines Suggest Molecular Tests
Issued in September 2007 to update a version released in January of that year, the NCCN guidelines explain that EGFR is found on the surface of epithelial cells in various cancers and mutations in certain areas of the EGFR gene that govern the protein’s activity. Certain EGFR mutations are significantly associated with positive response to TKIs, according to the guidelines.
Currently oncologists can order an immunohistochemistry test—which is not mentioned in the guidelines—and the two molecular EGFR tests. Mutation assays sequence the whole gene or certain parts using the polymerase chain reaction (PCR), while the second molecular assay determines how many copies of the gene are present in a tumor using fluorescence in-situ hybridization (FISH). Either is acceptable, according to NCCN, but the choice continues to be a matter of debate among oncologists and pathologists. Currently, Genzyme (Cambridge, Mass.) offers both molecular EGFR assays and K-ras tests, as do some academic cancer centers and reference labs.
K-Ras Mutation: Will TKIs Work?
The K-ras gene modulates cellular proliferation and differentiation. Found in 15–30% of lung adenocarcinomas, mutations are strongly associated with tobacco smoke exposure. “K-ras mutations are associated with intrinsic TKI resistance, and K–ras gene sequencing could be useful for the selection of patients as candidate for TKI therapy,” the guidelines maintain. For the patient, having a K-ras mutation is bad news because it’s unlikely he or she will respond favorably. Genzyme’s K-ras test uses a modified mini-sequencing platform to fluorescently detect genomic alterations at codons 12 and 13 in tumor specimens.
Ettinger usually orders K-ras and EGFR tests together, reasoning that “with a K-ras mutation, odds are the patient won’t respond to TKIs.” However, he’s not completely opposed to skipping both tests for patients with particular clinical characteristics. “If you’re limited in what’s available for second line therapy, if the patient has a limited smoking history and is female, you may just want to put them on TKIs. There’s a judgment call here,” he said. Others advise more stringent criteria for prescribing the drugs. “I wouldn’t deny TKIs if tests didn’t demonstrate an EGFR mutation, but I would deny them if a patient had a K-ras mutation,” explained Maureen Zakowski, MD, Attending Pathologist at Memorial Sloan Kettering Cancer Center (MSK) in New York, N.Y. and Professor of Pathology at Cornell University’s Weill Medical College. Mark Ladanyi, MD, Chief of the Molecular Diagnostic Service and Attending Pathologist at MSK, pointed out that K-ras and EGFR mutations never happen in the same tumor. MSK receives research funding from Genenetch and AstraZeneca (Wilmington, Del.), which also markets a TKI inhibitor.
While the guidelines and MSK’s treatment strategy both emphasize that a K-ras mutation probably means TKIs won’t benefit a patient, it’s not entirely clear that having the K-ras mutation automatically means TKIs won’t work, according to one researcher who has studied an EGFR assay that determines gene copy number and has received research funding from AstraZeneca, Genentech, and OSI Pharmaceuticals. According to Fred R. Hirsch, MD, PhD, Professor of Medicine and Pathology, University of Colorado Cancer Center in Denver, “The K-ras mutation occurs in twenty percent of unselected NSCLC patients. So you would think that eighty percent of patients would benefit from TKIs. But only about fifty percent of the patients do. That’s a gap of thirty percent, so I don’t think the K-ras story is completely understood. We’re working on identifying that thirty percent through prospective studies.”
The updated guidelines are already drawing increased attention to the EGFR and K-ras tests. Until now, Genzyme has seen slow but steady growth in demand for these assays, according to Bruce Horten, MD, Genzyme’s National Medical Director. “We’ve seen quite a bump up in demand since the NCCN guidelines were released,” he commented.
TKIs: Just One Now
Thus far, the FDA has approved two small-molecule drugs that target the tyrosine kinase activity of EGFR for patients with advanced NSCLC who haven’t responded to chemotherapy. Both erlotinib and gefitinib, marketed as Iressa (AstraZeneca, Wilmington, Del.), act as competitive inhibitors of ATP-binding at the active site of the EGFR kinase. However, following release of data from two failed clinical trials required by FDA as a condition of gefitinib’s 2003 approval, the drug was effectively removed from the market in 2005 via label language that stated the drug should be used only in patients who had experienced prior benefits. Erlotinib remains the only approved TKI inhibitor of EGFR, although several others are in development.
TKIs’ effectiveness is limited, however, because patients generally become resistant to them in less than a year, although some centers report that a few patients will take them for three years or more. Several studies are attempting to learn how resistance develops, with at least one gene fingered as the culprit (See Box, below).
Another Test Expected for Targeted NSCLC Therapy
In January, Genzyme announced an agreement with Moffitt Cancer Center in Tampa, Fla. that gives the company exclusive worldwide diagnostic testing rights to the discovery of two proteins that may help predict how patients respond to platinum drugs and gemcitabine, both of which are used to treat patients with NSCLC.
In its announcement, Genzyme said it would develop and market an assay that can be used to measure the expression levels of two proteins, ribonucleotide reductase and excision repair cross-complementation group 1, which are expressed by the genes RRM1 and ERCC1, respectively. Both proteins are involved in DNA synthesis and repair. The test would be used to guide first-line treatment for NSCLC patients and could help them avoid unnecessary side effects from ineffective treatment, the company added.
A Moffitt research team lead by Gerold Bepler, MD, PhD has shown that expression level of the two proteins correlates with patient response to platinum drugs and gemcitabine, Genzyme noted. At least one published paper from Bepler and his team strongly suggests that tumoral RRM1 expression is a major predictor of response to gemcitabine and platinum therapy, as is ERCC1 to a lesser extent (Journal of Clinical Oncology 2006; 24: 4731–4743).
In addition, a 2007 paper by Bepler and colleagues concludes that high expression of both RRM1 and ERCC1 is a determinant of survival after NSCLC surgery. (NEJM 2007; 356: 800–808). An accompanying editorial by Adi Gazdar, MD, deputy chief of the Hamon Center for Therapeutic Oncology and Professor of Pathology at the University of Texas Southwestern Medical Center in Dallas, notes that the two gene markers could affect selection of both those NSCLC patients with resected early-stage tumors who do not require adjuvant therapy and those patients who are not likely to benefit from conventional chemotherapy for advanced disease.
A Choice of EGFR Assays
There are two different methodologies to determine EGFR abnormalities in NSCLC. Mutation assays sequence the gene to allow examination of specific regions, namely exons 18–21. Exon 19 deletions are particularly good indicators of possible TKI response, the NCCN guidelines indicate.
Genzyme’s mutation test microdissects cells from specific tumor-rich areas, from which DNA is extracted and then amplified by PCR. Next comes bidirectional sequencing of exons 18 through 21 in the tyrosine kinase domain of the EGFR gene. Other labs offer partial analysis of the entire EGFR gene, as well as this examination.
While the NCCN guidelines recommend both the FISH and mutation assays, lab directors and recent review articles by researchers at Massachusetts General Hospital (MGH) and Harvard Medical School in Boston, Mass. note a debate as to which test is superior. The frequency of EGFR mutations in TKI-responsive cases is now well-established, according to authors of both articles, who noted that studies show that most patients who respond to TKIs have the mutations. But the articles also point out that certain research has also concluded that EGFR gene amplification, while not as predictive of response, may be equally as predictive of improved survival.
Ladanyi is a strong proponent of the mutation assay. He noted about 25 studies that confirm the link between mutations in EGFR and response to TKIs, pointing out that fewer have identified a link between copy number and such response. “Mutant EGFR can be amplified, but not always,” he explained. “The correlation between copy number and response is really due to EGFR amplified cases also being mutated. The key process is mutation. If they were separate processes, we would see cases with mutations where the other non-mutated copy of EGFR was also amplified.”
Ladanyi noted that recent research from MSK and other institutions, currently in press at Journal of Clinical Oncology, tested NSCLC tumors for both EGFR mutation and copy number and found lower response to TKIs among patients who had increased EGFR copy number but no mutations.
Noting practical concerns, Zakowski said she finds the mutation assay easier to use. “It’s the quickest, cleanest method. It tells you exactly what has mutated,” she explained. “FISH requires slides, it’s more time-consuming, and is subject to interpretation.”
A Case for FISH
Some evidence suggests that giving TKIs to patients with extra copies of EGFR can increase survival, even if these patients test negative for EGFR mutations and do not have an initial response. One such study by Hirsch and colleagues at University of Colorado Science Center and researchers at Italian centers looked at tumors from 102 NSCLC patients on gefitinib and tested them for EGFR status by FISH, DNA sequencing, and immunohistochemistry. Multivariate analysis showed that only high EGFR copy number, not mutations in the gene, were associated with better survival (HR 0.44; 95% CI 0.23–0.82) (Journal of the National Cancer Institute 2005; 97: 643–655).
According to Hirsch, a difference in the incidence of EGFR mutation—about 10% of NSCLC patients versus 35% to 40% of patients with extra copies of EGFR —means this method could potentially identify more patients who could benefit from TKIs. “Mutation is associated with a dramatic response to drugs, but is less so in Western populations,” he maintained. “The correlation of mutations with better survival is more clearly demonstrated in Asian populations. Future prospective studies might demonstrate survival benefit in mutation-positive patients. But which test is more appropriate when? Neither the FISH nor the mutation test is approved for diagnostic testing in clinical practice right now.”
Current mutation testing methods aren’t perfect, according to a recent editorial by Ladanyi and William Pao, also of MSK (Clinical Cancer Research 2007; 13: 4954–4955). Mutation tests must be quicker and more accurate before they become standard of care, they write. One problem is that some clinical samples do not have easily detectable levels of mutant EGFR. For a mutation to be detected by conventional direct sequencing, the tumor cells must comprise 25% of the total cells present in a given sample, they note. A lesser proportion could result in false-negative results after direct sequencing. Pao and Ladanyi point to more than a dozen recent papers reporting improved methods of detecting mutations.
Problems with EGFR Assay Studies
Retrospective analysis of clinical trials that aim to determine the predictive value of mutation and amplification assays have produced conflicting results in part from the studies’ different approaches, clinical designs, and variable interpretation of results, according to the Journal of Clinical Oncology review article.
The paper’s first author detailed a few of these problems. First, most trials of EGFR assays and TKIs are still retrospective, explained Lecia Sequist, MD, MPH, Instructor at Harvard Medical School and Assistant Physician at MGH. “Lots of randomized trials didn’t require tissue samples at the time of patient enrollment, and the molecular studies are based only on patients who happened to have tissue available post hoc,” Sequist added. As a result, different research teams have had various results. “There’s no clear theme to the publi-cations regarding EGFR gene amplification by FISH. Some studies have shown a benefit for TKI therapy in FISH-positive patients and others have not found one. It’s very interesting work, but it’s still shaking out.”
At least four prospective studies have used EGFR mutations as a method for selecting patients for TKI therapy, according to Sequist. She is lead author of one such study, currently in press at the Journal of Oncology. “We screened patients for EGFR mutations and treated only those that were positive with first-line gefitinib. We observed a response rate of fifty-five percent and a progression-free survival of 9.2 months. These results are two to three-fold of what is typically seen with standard chemotherapy in the first-line setting. While a randomized study needs to be performed for confirmation, the initial results with this strategy are promising.”
Hirsch hopes that research by the FDA’s Critical Path Initiative (CPI), which aims to speed the development and evaluation of cancer therapies, will lead to an FDA-approved EGFR test for NSCLC. The CPI’s Oncology Biomarker Qualification Initiative, which was launched in February 2006 and includes members from FDA, NCI, and CMS, is planning a clinical trial that will directly compare predictive value of protein expression, mutation, and FISH EGFR assays, according to Maryellen de Mars, PhD, Director for Clinical Biomarkers in the Rockville, Md. office of the Critical Path Institute. Her nonprofit organization is coordinating the effort for the federal agencies.
The Test of the Future
Proponents of both the mutation and FISH assays agree that decisions about treating patients with EGFR mutations are unlikely to rely upon one particular kind of assay or marker in coming years. “It may be that the diagnostic paradigm should be a combination of tests because nothing by itself is 100% perfect,” Hirsch remarked. “My sense is that mutation assays are definitely important and give lots of information. FISH is probably important too, and together they may be more so,” added Sequist. “These cancers are complex. No one test gives you the golden answer. There probably will be a panel eventually and lots of centers, including Massachusetts General Hospital, are working on them.”
For More Information
The following are useful sources of information on tests to detect EGFR and K-ras abnormalities:
The National Comprehensive Cancer Network’s Non-Small Cell Lung Cancer (NSCLC) Guidelines are available at the NCCN Website.
A review article by Daniel Haber, MD, Lecia Sequist, MD and their colleagues at Massachusetts General Hospital summarizes the biology of EGFR in NSCLC, the clinical and molecular predictors of whom will benefit from EGFR tyrosine kinase inhibitors, use of patient-specific molecular profiling, and future directions of related research (Journal of Clinical Oncology 2007; 25: 587–595).
Another review article by Haber and colleagues summarizes the results of genetic, biochemical, and clinical studies focused on somatic mutations of EGFR that are associated with TKI’s effectiveness. The review also explains how understanding the genetic heterogeneity of epithelial tumors and devising strategies to prevent their resistance to TKIs are essential to successful use of the agents (Nature Reviews Cancer 2007; 7: 169–181).