NEW YORK (GenomeWeb) – By examining and re-examining which gene mutations were present in a patient's tumor, clinicians were able to adjust treatment as the patient's lung cancer progressed.
In the New England Journal of Medicine this week, researchers from the Massachusetts General Hospital Cancer Center and elsewhere recounted the case of a woman with metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer. She was first treated with Pfizer's crizotinib, an ALK inhibitor, but soon developed resistance to the drug, resistance that they traced to an ALK C1156Y mutation using a targeted sequencing-based PCR assay. After responding to lorlatinib, a next-generation ALK inhibitor being developed by Pfizer, the patient was switched backed to crizotinib when her metastases reappeared, as new mutations detected through sequencing made the tumor sensitive to the drug again.
"These results highlight how important it is to obtain repeat biopsies in patients who relapse on targeted therapies," first author Alice Shaw, an associate professor of medicine at Harvard Medical School, said in a statement. "Molecular profiling of these biopsies can uncover novel mechanisms of resistance. In some cases, this information can then help us to select the next therapy that's most likely to be effective."
She and her colleagues obtained biopsies from the lung cancer patient at different time points as her disease progressed. Initially, a fluorescence in situ hybridization assay conducted on a biopsy from a liver lesion showed ALK rearrangements, though no ALK amplifications and no MET amplification. In later samples, a targeted next-generation sequencing assay uncovered two mutations in the ALK kinase domain: C1156Y and L1198F. No other mutations were present in any of the 38 other cancer-linked genes the researchers analyzed.
ALK C1156Y, the researchers said, is a known crizotinib-resistance mutation, while ALK L1198F has been reported to be a gain-of-function mutation in anaplastic thyroid cancer and a brigatinib-resistance mutation when combined with F1174V in a nucleophosmin-ALK-rearranged cell line.
The two ALK mutations were present at similar frequencies in the woman's tumor, suggesting they were present on the same allele of the ALK fusion gene. The researchers confirmed their suspicion by amplifying EML4-ALK from a frozen tissue specimen, sub-cloning the PCR products into a vector, and sequencing resulting bacterial colonies.
Shaw and her colleagues also performed exome sequencing on tumor tissue samples collected from the patient before treatment, after relapse while on crizotinib, and after her second relapse while on lorlatinib.
The ALK C1156Y mutation was present at low levels in the pre-treatment sample, they reported, as well as in the crizotinib-resistant sample. The double ALK C1156Y-L1198F mutation, meanwhile, was only in the lorlatinib-resistant sample.
The number of tumor cells harboring the ALK C1156Y mutation increased from less than 7 percent in the pre-treatment sample to about half in the crizotinib-resistant sample and then to 100 percent of the lorlatinib-resistant sample, they added.
This and clonal analyses indicated to the researchers that a minor subclone carrying the ALK C1156Y mutation was present even before crizotinib treatment began, was enriched during treatment, and further acquired an ALK L1198F mutation as the patient's disease progressed.
After relapsing on lorlatinib, the patient was given crizotinib a second time and responded to it before again relapsing. Targeted NGS testing of a biopsy obtained at that second relapse while on crizotinib indicated that the ALK L1198F mutation was no longer present. This, the researchers said, is consistent with the suppression of the C1156Y-L1198F subclone by crizotinib.
To check that ALK C1156Y-L1198F does indeed mediate lorlatinib resistance, Shaw and her colleagues generated cell lines expressing ALK C1156Y, ALK L1198F, or the ALK C1156Y-L1198F double mutation. Cell survival assays showed ALK C1156Y-L1198F was resistant to lorlatinib as well as second-generation ALK inhibitors.
Crizotinib, they noted, inhibited both ALK C1156Y-L1198F and wild-type ALK with similar potency.
ALK L1198F was sensitive to next-generation ALK inhibitors including lorlatinib, and ALK C1156Y-L1198F phosphorylation was suppressed by crizotinib, but not by lorlatinib. This, the researchers said, corroborates observations made in the clinic that C1156Y-L1198F causes lorlatinib resistance, but also sensitivity to crizotinib.
In other mutants the researchers generated, they found that the addition of L1198F increased crizotinib sensitivity and resistance to other ALK inhibitors.
Binding affinity studies further found that the L1198F and C1156Y-L1198F ALK mutants had decreasing binding affinity for lorlatinib and other next-generation ALK inhibitors, but increased affinity for crizotinib.
"For patients relapsing on first-generation inhibitors like crizotinib, treatment with more potent and selective next-generation inhibitors can be very effective," Shaw said. "However, cancers that become resistant to next-generation inhibitors are usually also resistant to less potent first-generation inhibitors. It caught us by surprise to discover a mutation that could cause both resistance to a newer next-generation inhibitor and re-sensitization to the older, first-generation inhibitor."