A Massachusetts General Hospital-led study has demonstrated the feasibility of routine clinical genotyping of patients with non-small-cell lung cancer for alterations in more than a dozen genes, and suggests the approach can direct personalized treatment more comprehensively than single-gene tests.
Using a PCR-based assay called SNaPshot targeting about 50 known mutations in more than a dozen genes, the researchers prospectively analyzed samples from several hundred NSCLC patients seen at MGH and the affiliated North Shore Cancer Center over approximately one year.
In their study, which appeared online in the Annals of Oncology Nov. 9, the team found that more than half of the samples they genotyped with SNaPshot had one or more gene alterations. For almost 100 patients with advanced disease, the mutation information allowed the group to provide or recommend targeted treatments, Lecia Sequist, a thoracic medical oncologist at MGH and a co-lead author of the study, told PGx Reporter.
"The main take-home point, especially for the practicing doctor out there, is that genotyping is becoming really important in the care of lung cancer patients," Sequist said.
"What we tried to show in our paper is that it can be done and integrated into a busy clinic today, and also that it can actually influence patient care, too," she added. "It's not just some esoteric academic exercise; it can actually influence the way you treat patients."
While testing for some of the most common genes implicated in NSCLC, such as EGFR and KRAS, has been used to determine patients' suitability for certain targeted drugs for some time, less common mutations also confer potential benefit from new pharmacogenomic drugs.
Testing for mutations in a larger number of genes would be impractical if done in a stepwise manner, because of the large sample sizes needed, according to Dora Dias-Santagata, also a lead author on the study and co-director of MGH's Translational Research Laboratory. Santagata told PGx Reporter that the group's SNaPshot assay was developed as a way to interrogate a larger swath of mutations more efficiently, focusing on associating these SNPs to marketed and investigational targeted drugs.
"Especially with lung cancer, sometimes you get these very small biopsies," she said. "So you want to try to maximize the amount of information you can extract from a small amount of DNA you're going to get from this tumor tissue."
Since completing the study, MGH has joined the 14-hospital Lung Cancer Mutation Consortium, within which several other lung cancer genotyping efforts have begun, using a variety of platforms.
Santagata's lab created their SNaPshot multiplex assay using the Applied Biosystems' SNaPshot PCR and electrophoresis platform. The version used in the group's NSCLC study included more than 50 mutations in 14 genes. Now in its third version, the panel has since expanded to around 70 mutations, Santagata said, as the group has added new genes to expand the assay to address different cancer types or additional targeted treatments.
"We were trying to focus on [genes] for which there were targeted therapies available already … to try to capture genetic aberrations that could be used by clinicians to then gear their patients to the best available targeted therapies," she said. The platform, she explained, allows the group to easily expand and change the panel of genes and mutations over time.
In its NSCLC study, the group used SNaPshot to genotype patients who were seen at Mass General Hospital and the North Shore Cancer Center between March 2009 and May 2010. The authors reported that when the program was initiated, only patients with adenocarcinoma were eligible for testing, but by August 2009 they could include all NSCLC patients.
The researchers referred 589 patients for genotyping during the trial period, from which 552 samples had enough material for testing. The group successfully genotyped the 552 samples from 546 patients, profiling about 80 percent with the first version of the SNaPshot assay, and another 20 percent with the second version, which included additional mutations in AKT1, HER2, and IDH1.
The group also tested almost all the samples using FISH for ALK alterations. On average, testing took an average of 2.8 weeks from a sample being sent for testing to final results, the team reported.
Fifty-one percent of the samples showed at least one mutation on the SNaPshot panel and/or an ALK translocation, 25 samples were positive for two mutations, and two tumor samples had three simultaneous mutations, the authors reported.
With continued expansion to the SNPs interrogated by SNaPshot, Sequist hopes the test will be able to identify even more patients harboring a mutation.
The majority of alterations were in the more commonly implicated genes, although the team turned up several cases of rare, but still pharmacogenomically actionable markers. Overall, there were 73 EGFR mutations, 134 KRAS mutations, 27 ALK translocations, 26 TP52 mutations, 22 mutations in PIK3CA, 11 mutations in B-catenin, nine in BRAF, six in NRAS, two HER2 mutations, and one in IDH1.
"One of the advantages of having a system like this … is that you do manage to capture the small percentage of patients with mutations in the genes that are not the most common genes affected," Santagata noted.
Incorporating SNaPshot into a hospital setting like MGH could have a broad impact on patient care. A few years ago, patients with lung cancer coming for treatment at MGH would have been tested just for KRAS and EGFR, according to Santagata.
"But if you happen to be part of this small slice of four percent of PIK3CA-mutation patients, you wouldn't even know you could potentially [benefit] from being treated with a PIK3CA inhibitor," she said. "I think it's important to have a comprehensive test that's going to give you not only what you expect to see, but also the less frequent occurrences that for that individual patient are going to be important."
SNapShot can also be a tool for matching patients to clinical trials investigating PGx drugs, for which it is often hard to recruit participants, particularly if the disease population is small and the biomarker is rare.
"Within lung cancer," Sequist said. "BRAF mutations are not that common, and in our study we found only nine patients." However, she added that a small group was able to go on to clinical trials of BRAF-targeting drugs like Roche/Plexxikon's Zelboraf. That drug, indicated for metastatic melanoma patients harboring BRAF mutations, made waves earlier this year with its clinical success and FDA approval (PGx Reporter 8/17/2011)
"Even though it was a rare mutation, lung cancer unfortunately is so common that even a small slice of the pie is still a quite large number of people," she said. "There are probably more people with BRAF-mutant lung cancer than there are with gall bladder cancer or some other types total, so it definitely can make a splash."
In the study, 353 subjects had advanced disease, the researchers reported, and of these, 170 had a mutation or translocation in EGFR, KRAS, ALK, BRAF, PIK3CA, or HER2 that the study authors deemed "potentially targetable." About forty percent of these, or 64 patients, went on to enroll in at least one trial using a targeted therapy designed to block one of these genes or a related downstream pathway impacting driver mutation signaling, the authors wrote.
Additionally 30 more EGFR-mutant subjects went on to "off protocol" treatment with erlotinib, an EGFR inhibitor marketed by Genentech as Tarceva, due to genotyping results. This means that overall about 22 percent of the advanced NSCLC patients underwent some type of genotype-directed targeted treatment.
The authors wrote that it was not possible to measure how many patients were directed away from possible treatments that they were unlikely to respond to due to genotyping results though they said they suspect that this also occurred.
According to Santagata, the SNaPshot panel is currently being expanded to a fourth version, and MGH is now using it to profile a variety of solid tumors in addition to lung cancer. The group is also working to adapt the test to hematologic cancers, such as leukemia.
Eventually, she said, because of the heterogeneity of diseases like lung cancer, it would be beneficial to transition to using deep sequencing technologies instead of genotyping to profile patients' tumors more precisely. In the near term, however, Santagata felt that the SNaPshot approach was more likely to be adopted by other centers since the multiplex genotyping platform is more feasible and cheaper than deep sequencing.
Santagata's team has applied for a patent on the SNaPshot tumor genotyping assay and has licensed the technology to Bioreference Laboratories. According to Santagata, who is a consultant for Bioreference, the company is preparing to offer this "as a commercially available test."
Along with MGH, some other partners in the Lung Cancer Mutation Consortium are using SNaPshot to explore different strategies in mutation analysis. Santagata said her group worked with another team at Vanderbilt University to adapt SNaPshot to their purposes.
"Their decision was slightly different. They decided to make panels that would be tumor-specific, so [they created] one panel for lung, one for melanoma, one for colorectal," she said, whereas MGH's assay uses a larger panel applicable to a variety of cancers.
Meanwhile, Memorial Sloan-Kettering Cancer Center has taken a mass spectrometry-based approach, using Sequenom's MassArray genotyping system to do similar multiplex analysis of NSCLC patients.
Mark Ladanyi, a pathologist in the Human Oncology and Pathogenesis Program at MSKCC, discussed the center's early work with the Sequenom panel at the Next Generation Dx Summit in Washington, DC, in August. At first, the MSKCC project was performing single-gene tests for EGFR and KRAS, and following them with the Sequenome panel, but he told PGx Reporter this week that the center now uses the multiplex genotyping right off the bat.
While MGH is broadening its panel for a large variety of cancers, MSKCC has focused its assay on lung and colorectal cancers, deciding to include more mutations in EGFR, KRAS, and BRAF than the MGH panel, while leaving out other gene mutations. Having a narrower disease focus when there are small biopsy sample sizes, such as in lung caner patients, may be a better approach in some cases, according to Ladanyi.
"It's certainly simpler in terms of workflow and ordering to just have one panel for everything," he said, "[But] in some small biopsies, even with these highly efficient genotyping technologies, you may start to hit [a point where] the sample may not be enough for all the genotyping you want to do."
"You don't necessarily want to waste your DNA on genes that are extremely unlikely to be mutated in that type of cancer," he said.
But Ladanvi said the two approaches are more similar than they are different, and observed that clinician engagement facilitated by the consortium has helped accelerate multiplex genotyping approaches in cancer, among other partners in the Lung Cancer Mutation Consortium.
While several centers came into the consortium with comprehensive NSCLC genotyping efforts already underway, many took up comprehensive genotyping approaches only after they joined the collaboration. Both the Sequenom and SNaPshot approaches are also being used by other members, Ladanyi said.
Sequist predicted that for NSCLC, most clinical groups will move in the future toward using a broad "platform of testing" rather than testing each gene one by one.
"It can be very hard to predict which test is the most important for a certain patient," she said. "The more mutations we have with targeted drugs, some are more common in smokers or non-smokers, different genders … It can become impossibly tricky for any doctor to figure [which test to order] for their patient."
Moving forward from the lung cancer study, Sequist's group now plans to track how patients are faring on the targeted treatments clinicians have prescribed them based on genetic information.
"Most of these drugs work for a period of time and then stop as the cancer becomes resistant," Sequist said. "We are trying to make this an iterative process, so the first time they get diagnosed we find out what the genetics are and try to personalize treatment. But when it wears off, we do the same cycle again and look at the genetic landscape at that time and see what options would be best."
Currently, it's unclear how the genetically guided targeted treatments given to patients actually affect their outcome and survival.
"It's hard to answer … because we don't have enough follow-up information. We've only been doing this testing for a little over two years," Sequist said. "Nowadays, many lung cancer patients live for two years, so we just don't know how long people are surviving."
She said it's realistic to think that being able to personalize treatment based on genomic data should positively impact survival, but direct studies will be needed to show this.
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