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Phosphoproteomics Study Drives Phase I Trial Testing Pfizer ALK Inhibitor as Breast Cancer Therapy


By Adam Bonislawski

Researchers at the University of Texas MD Anderson Cancer Center and George Mason University presented data this week that suggests anaplastic lymphoma kinase, or ALK, inhibitors may be useful in the treatment of inflammatory breast cancer.

Via a phosphoproteomic analysis of tumor samples using reverse phase protein arrays, the scientists linked IBC to heightened ALK signaling and ALK gene amplification. Based on these results, they have begun a phase I, dose-escalation clinical trial testing the use of the ALK inhibitor crizotinib – sold by Pfizer as Xalkori – in IBC patients. The drug is currently approved for use in locally advanced or metastatic ALK-positive non-small cell lung cancer.

The study was led by MD Anderson researcher Fredika Robertson, with Emanuel Petricoin and Lance Liotta — co-directors of GMU’s Center for Applied Proteomics and Molecular Medicine – heading the RPPA phosphoproteomics work. The researchers presented data from the study at this week's American Association for Cancer Research-National Cancer Institute-European Organization for Research and Treatment of Cancer International Conference in San Francisco.

The collaboration, Robertson told ProteoMonitor, stemmed from a meeting between the researchers at a grant review session for the Susan G. Komen Foundation. Robertson was able to provide access to resources including IBC patient samples, mouse models, and cell lines, while Petricoin and Liotta brought their expertise in the RPPA technology they helped develop.

RPPAs use cell lysates spotted in array formats that can then be probed with antibodies to multiple proteins of interest. This enables researchers to simultaneously measure expression levels of numerous analytes, which can be helpful in identifying protein pathways that exhibit altered activation in disease samples.

“We sent them samples, and [Petricoin] started doing the reverse phase proteomic analysis,” Robertson said. “About a year ago, [Petricoin] called me, and he said, ‘You won’t believe the data we got from this first set of samples – this ALK pathway is activated.’”

“That was completely unexpected, and so that was when we really started in earnest to believe that this was something worth pursuing,” she said.

“We were stunned when we saw the ALK pathway just light up,” Petricoin told ProteoMonitor. "ALK activation and downstream signaling [was heightened] in almost every one of the IBC samples that we looked at, and in none of the non-IBC samples. It was just evident when we looked at the data that something was going on.”

Based on these findings, the researchers next began to investigate whether IBC tumors would respond to treatment with the ALK inhibitor crizotinib. Using xenograph mouse models, they found that “these tumors were tremendously sensitive to crizotinib,” Petricoin said.

“These are mouse xenographs that are highly resistant to just about everything, so the fact that these tumors melted away [when treated with crizotinib] provided further evidence that what we were seeing had some functional consequences,” he said.

ALK abnormalities are associated with diseases like non-small cell lung cancer and lymphoma but hadn’t previously been tied to IBC. IBC tumors are largely HER2 positive. However, patients typically develop resistance very quickly to agents targeting HER2, and diagnosis of the disease carries a five-year survival rate of only 40 percent.

Based on the RPPA work and the success of crizotinib in the IBC mouse models, the scientists investigated the disease’s genetic underpinnings and found that 13 out of 15, or roughly 87 percent, of the IBC patients they examined had amplification of the ALK gene.

This amplification would likely have been missed had the researchers relied solely on a genomic method, Petricoin noted. “A genomic analysis probably would have missed it until someone did a full genomic sequencing where they would see the amplification,” he said. “If someone had gone in and surveyed other tumors with crizotinib sensitivity, they would have done a translational analysis and a mutational analysis, and they would have missed [the amplification].”

Robertson said her interest in applying proteomics to IBC stemmed from other breast cancer research – particularly in the realm of HER2-driven treatment – that suggests that “while genomics can give us some information, we really have to look to the protein level.”

According to Robertson, recent proteomics work has shown that even triple-negative breast cancer patients – whose tumors don’t express HER2, estrogen receptor, or progesterone receptor – exhibit heterogeneity in HER2 expression.

“There’s data that’s emerging now that shows HER2 drives survival of cancer stem cells of the breast,” she said. “So even in triple-negative disease, [HER2] might not be amplified but is present in a small number of cells, and those cells are believed to be what initiates and sustains the tumor. So there’s the suggestion that even a patient that is not HER2 amplified may benefit from treatment with HER2 [inhibitors].”

“[Many] companion diagnostics have been based on genomics, and we’re seeing that that’s not going to be sufficient, really, to inform us about patient selection or about the mechanisms of drugs,” she added.

In collaboration with Massimo Cristofanilli, chair of medical oncology at Fox Chase Cancer Center, Robertson has launched a phase I clinical trial testing crizotinib in IBC patients exhibiting ALK activation.

Along with Liotta and Petricoin, she is also investigating whether normal cells manipulated to overexpress the ALK gene become tumor generating. Early data suggest “that that is absolutely the case,” she said, adding that if these initial results hold they plan to present these findings at upcoming meeting.

Petricoin said that if crizotinib proves effective in treating ALK-positive IBC, the RPPA ALK assay could be commercialized as a companion diagnostic by Theranostics Health, a biotech firm he and Liotta founded to market the RPPA technology.

Theranostics is “working on developing an overarching menu of assays for physicians to order, and [the ALK assay] could be part of their menu,” he said. Testing might also be done via an fluorescence in situ hybridization assay to detect ALK amplification. In August the US Food and Drug Administration cleared Abbott’s Vysis ALK Break Apart FISH Probe for use with crizotinib (GWDN 8/29/2011).

Alternately, if ALK amplification broadly exhibits the almost 90-percent penetration in IBC patients that it demonstrated in the MD Anderson-GMU study, a companion diagnostic might not be needed at all, Petricoin suggested.

“If the incidence rate [of ALK amplification] really is that high, you might not need any test to prestratify,” he said. “It could be that you just treat any patient with progressing IBC with crizotinib.”

Petricoin cautioned that crizotinib was unlikely to be a magic bullet for the disease, noting that just as IBC patients typically develop resistance to HER2-targeted therapeutics, “it’s very likely that they will develop resistance to crizotinib.”

“We may need to partner crizotinib with other agents targeting downstream signaling,” he said. “Maybe that would be the killer application.”

And, of course, it remains to be seen what the effects will be in actual patients, he added, noting that “if it doesn’t work clinically it just becomes another academic finding.”

Nonetheless, Petricoin said, the work offers a “great case study” for the power of proteomic assays in the companion diagnostic and drug repurposing arena.

“Here you have a proteomic test that found ALK activated. There’s a drug on the market for ALK. And we can test this hypothesis right away in patients who have progressed off the standard of care,” he said.

"I think it’s a lesson for people who are considering using just genomic platforms for personalized medicine,” he added. “There’s a lot of value in direct protein measurement, especially when the drug is being directed against a protein.”

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.