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Researchers Test Multiplex Genotyping of Lung Cancer Samples in Clinical Setting

NEW YORK (GenomeWeb News) – A team of researchers from Massachusetts General Hospital, Harvard Medical School, and Yale University has demonstrated the feasibility of routinely genotyping lung cancer samples for alterations in more than a dozen suspicious genes in a clinical setting.

Using a multiplex PCR-based method called SNaPshot, the researchers assessed hundreds of non-small-cell lung cancer samples as part of a clinical testing regimen, looking for mutations at more than 50 sites in 14 genes implicated in past studies of the disease. The same samples were also tested by fluorescence in situ hybridization to look for translocations affecting another NSCLC-associated gene called ALK.

In their study, which appeared online in the Annals of Oncology, the team found that some mutations tended to appear together or to coincide with certain clinical characteristics or outcomes. And, they reported, incorporating the genetic information helped to provide more targeted treatment for some of the patients who had the most advanced cancers.

"Because each tumor will harbor a specific set of mutations, the SNaPshot test allows us to match individual patients with the therapies that will most likely be effective in treating their cancer," co-corresponding author Dora Dias-Santagata, a Harvard Medical School and Massachusetts General Hospital pathology researcher, who also co-directs the Massachusetts General translational research lab, said in a statement.

Genetic studies have been uncovering genes and pathways that tend to be altered in one or more cancer sub-types, including some that may be candidates for targeted treatment, the researchers explained. Even so, they added, it can be difficult to quickly test for multiple mutations.

For the current study, Dias-Santagata and colleagues came up with a clinical genotyping test based on Applied Biosystems' multiplex PCR-based SNaPshot system to efficiently genotype NSCLC tumors at more than 50 sites in 14 candidate genes.

Using the SNaPshot approach, they attempted to genotype formalin-fixed, paraffin-embedded NSCLC samples collected from 589 NSCLC patients as part of routine clinical testing. Along with this genotyping, they also used FISH to look for translocations involving the ALK.

Although they did not have a sufficient amount of tumor tissue available to test for some of the patients, they managed to get multiplex genotyping data for 552 samples from 546 of the individuals. The turnaround time per sample ranged from one week to 8.9 weeks, with a median time of 2.8 weeks.

Just over half of the tumors — 51 percent — contained mutations in one or more of the genes tested. The most commonly affected genes were KRAS and EGFR: the researchers found cancer-related changes at the KRAS variants tested in nearly a quarter of the tumors and EGFR mutations in 13 percent.

Mutations in PIK3CA and TP53 turned up in 4 percent and 5 percent of samples, respectively, they reported, and ALK translocations were detected in 5 percent of the tumors. Several other genes contained less frequent mutations.

"In contrast with prior genotyping strategies (mainly focused on testing EGFR and KRAS), employing a broad gene panel enabled us to provide a therapeutic alternative to lung cancer patients whose tumors harbored much less frequent genetic abnormalities," Dias-Santagata said.

Moreover, the team found that the presence of certain mutations in the lung tumors often corresponded to certain clinical characteristics. For example, individuals with squamous cell lung cancers also tended to have alterations affecting the PIK3CA gene. Those who had a history of heavy smoking, meanwhile, more often had tumors harboring KRAS and NRAS mutations.

When they focused on the 353 patients with the most advanced lung cancers or who had had cancer recurrence, the team found that 48 percent carried mutations in genes for which targeted treatments were potentially available. And 22 percent of the advanced lung cancer patients enrolled in a targeted therapy trial coinciding with their genotype profiles.

Based on their findings, the researchers concluded, "Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice."

The same approach is expected to be useful for other cancer types as well. The researchers are currently testing similar multiplex genotyping approaches in breast cancer, colorectal cancer, and gliomas and intend to look at blood cancers such as acute myeloid leukemia in the future.

"We hope that this approach will help us identify therapeutic options for a much larger fraction of cancer patients and provide a good resource to understand differences in response to therapy," Dias-Santagata said.

Even so, those involved in the study cautioned that the multiplex genotyping method appears to be better suited for finding mutations in oncogenes than tumor suppressor genes, perhaps because oncogenes tend to harbor a more limited set of changes. In the current study, for instance, researchers found mutations in the tumor suppressor coding gene TP53 in just 5 percent of tumors tested — fewer than expected based on previous studies of NSCLC.

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