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Pfizer and Collaborators Developing Next-Gen Sequencing Test for Crizotinib Resistance Mutations

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Researchers from Pfizer and collaborators in Korea and Greece are researching a testing method to identify mutations in patients who develop resistance to Pfizer's Xalkori (crizotinib) and may benefit — depending on their mutation status — from treatment with a second-generation ALK inhibitor or other treatment strategies.

The group published a description of the method this month in the journal Genomics, using the Ion Torrent PGM to deep sequence the ALK kinase domain of tumor DNA and using a base-specific calling algorithm to detect previously identified resistance mutations in a small group of patients who relapsed after treatment with crizotinib.

Members of the same group, led by Pfizer researcher Mao Mao, have also been working to develop alternative testing methods to detect which patients have ALK fusions and thus could benefit from crizotinib with better sensitivity and efficiency (PGx Reporter 12/19/2012).

ALK fusions conferring sensitivity to crizotinib are present in about 3 percent to 5 percent of patients with non-small cell lung cancer. But many of these patients with initial encouraging response to the drug later develop resistance. About a third of these cases are due to secondary mutations in the ALK tyrosine kinase domain, according to the study authors.

In the new study, the group reported that in a 13-patient cohort, the single-base-calling sequencing method was able to detect three previously reported ALK kinase resistance mutations in three patients.

According to the authors, the approach could help in the clinical development of second-generation ALK inhibitors being developed by Pfizer and others.

It could also, as these drugs enter the market, help doctors choose which treatment to prescribe based on each drug's potency toward specific ALK kinase resistance mutations, according to the study, or help decide when to use alternative kinase inhibitors or combination therapies.

Potentially, the testing could even serve as a direct companion diagnostic to some of these second-generation therapies with specific potency toward one or another mutation, though Pfizer has not said that the research study is progressing toward any specific companion diagnostic goals.

The company did say in a statement to PGx Reporter that “Pfizer continues to work with investigators to evaluate Xalkori and better understand potential mechanisms of resistance. The identification of resistant mutations to Xalkori adds to our growing body of knowledge around the selective inhibition of the ALK fusion gene and the treatment of this distinct subset of patients.”

To develop the sequencing method the Pfizer group used for its recent study, Mao and his colleagues first used DNA from a crizotinib-resistant cell line and a series of titration experiments to determine the sensitivity of the PGM assay. According to the study authors, the method should be able to detect the mutation above 0.8 percent with 99 percent confidence.

Then the team applied the assay to DNA samples from 13 patients who relapsed after treatment with crizotinib. To increase the accuracy of the assay, instead of using a noise cutoff based on average error across the sequence data, the group assessed noise levels at each base pair using an algorithm the group calls BASCA.

The team was able to identify three different mutations with an allele frequency between 12 percent and 15 percent in three patients among the cohort of 13. Each — C1156Y, L1196M, and G1269A — had been previously identified as present in crizotinib-refractory patients.

In the study the group also successfully amplified DNA for sequencing from a variety of sample types, including FFPE tissue, pleural fluid, and bronchoscopic biopsy, supporting the clinical potential of the approach in NSCLC where the availability of sufficient tumor tissue for testing without repeat biopsy is an issue, the authors wrote.

According to the group, the entire testing process — analyzing 8 to 16 samples per sequencing run — can be completed in three working days. This suggests the assay could be used to make clinical decisions quickly when patients show newly acquired resistance to crizotinib.

In the paper, Mao and his colleagues wrote that the group is planning to continue testing the sequencing method using additional clinical samples collected from relapsed patients.

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