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Proximity Ligation Assays Show Potential to Detect Altered EGFR Signaling, Predict Drug Response

PLA image

NEW YORK (GenomeWeb) – A study by a team from the Moffitt Cancer Centerhas demonstrated that proximity ligation assays, which detect aberrant gene signaling, can predict response to EGFR-inhibiting drugs.

The study results, published this week in Science Signaling, suggest that this approach could potentially complement mutation-based molecular diagnostics currently used to guide treatment with EGFR inhibitors, by detecting targetable aberrant EGFR signaling even in patients who have normal genotypes.

Eric Haura, the study's senior author, told GenomeWeb that he and his colleagues have for some time been using mass spectrometry and other tools to investigate how genes and proteins interact in the cellular environment, including in-depth analyses of how expression of EGFR is mediated by signaling-associated proteins.

The group then became interested in how measuring these signaling interactions might be useful in clinical practice, and turned to proximity ligation assays as a way to measure some of the same cellular processes, but in a way that could be applicable to clinical practice. PLAs detect the presence of target proteins by creating a detectable amplicon when two complementary tags ligate as the molecules come into proximity with each other.

"We realized that proximity ligation assays could give us a way to take that knowledge from these discovery studies and develop assays that can then go look for specific complexes in human disease samples," Haura said.

In their study, he and his colleagues described the development of a PLA to detect the interaction of EGFR with its major signaling adaptor protein, growth factor receptor-bound protein 2 (GRB2). The team used multiple cell lines to show that levels of EGFR-GRB2 interaction correlated with established biochemical measures of EGFR signaling and with sensitivity to EGFR inhibiting drugs.

The group then tested the assay to see how results matched up with drug response in human tumors. First, they looked at how the level of EGFR-GRB2 interaction matched with the shrinking of tumors implanted into mice that were treated with EGFR-inhibiting drugs.

Initially, the group compared two mouse populations, one grafted with a tumor carrying an EGFR activating mutation and one without. PLA analysis revealed strong activity in the EGFR-activated xenograft, which then died down significantly after treatment. In contrast, the non-EGFR mutated tumor showed a much less intense EGFR-GRB2 PLA signal, and also minimal change in response to the drug.

To expand this, the researchers then performed the PLA on a tumor microarray containing samples from 291 different mouse xenograft tumors, comparing PLA with a direct measurement of protein levels to establish whether the method offered additional information over this simpler measurement of gene signaling.

Focusing down on only lung cancer xenografts in the array, the group segregated samples by their measured levels of EGFR-GRB2 proximity into low- and high-PLA groups. They found that tumors with high EGFR protein abundance were more likely to also have high EGFR-GRB2 proximity, while those with intermediate abundance mapped to both the high- and low-PLA groups.

In cetuximab-treated lung cancer xenografts, PLA did not predict drug response overall, but the researchers did notice that four out of six drug-responsive mice with contradictory wild-type EGFR genotypes did have high EGFR-GRB2 PLA scores.

The team then moved on to look at how PLA score in stored samples from 350 patients with different stages, molecular etiologies, and pathological subtypes of non-small cell lung cancer correlated with EGFR signaling. Looking at this collection of three cohorts, the group found that patients with high PLA scores also showed high EGFR signaling.

More importantly, PLA results did not always correlate with genomic EGFR status. For example, 30 percent of tumors without expected mutations still showed high PLA scores.

Looking further, the team found that a significant number of these patients with wild-type EGFR also saw benefit from anti-EGFR therapy, indicating that using EGFR-GRB2 PLA might be a way to pick out drug responders who would not otherwise be identified using current standard of care gene-mutation tests.

In 91 patients with known clinical outcome data who received either erlotinib or gefitinib, the team found that high EGFR-GRB2 PLA was significantly associated with improved overall survival.

According to the authors, the results provide initial evidence that detecting signaling-associated protein complexes using proximity ligation assays has the potential, if further validated, to guide therapeutic decisions.

Highly active EGFR signaling, as measured by higher levels of EGFR-GRB2 proximity, was not limited to tumors with activating EGFR mutations in the study, but was also present in wild-type cancers. Moreover, tumors with high EGFR-GRB2 PLA were overall more likely to respond to anti-EGFR therapy, although this signaling was not entirely predictive of response.

Before pursuing the technique further for clinical use, Haura said, the group will have to replicate its findings in additional clinical samples — something it is currently working to do.

"What we have is very encouraging but we need more work to make this come to fruition," Haura said." The group is also weighing whether this work would be better pursued in the academic or commercial setting.

Promisingly, measuring signaling-associated protein complexes via proximity ligation is not limited to EGFR. The group is also actively investigating MET and FGFR, Haura added.

With MET, he said, the team is also interested in developing a multiplex system to evaluate PLA levels of both EGFR and MET simultaneously, and has completed some early pilot experiments in this vein.

Even further multiplexing is also on the table. "You could almost imagine building whole pathways in multiplex, where … you can actually march along … and walk your way down to show the true connection of all the proteins functioning in a pathway," Haura said.