Proteome Sciences has published the first peer-reviewed study of its Sysquant phosphorylation assay, using the product to investigate signaling pathways in pancreatic cancer.
Detailed in a paper out last week in PLOS One, the study used the company's mass spec-based Sysquant assay to profile global phosphorylation patterns in 12 resected pancreatic ductal adenocarcinomas, identifying a number of dysregulated phosphoproteins and signaling pathways, including several known drug targets.
In total, the researchers identified 2,101 proteins, 152 of which showed significant differences in abundance between tumor and non-tumor tissue. They also identified 6,542 unique phosphopeptides, 635 of which showed significant regulation.
While the authors noted that, given the large amount of data generated by the Sysquant assay, their analyses were not exhaustive, they identified as significantly dysregulated in the tumor samples pathways involved in cell migration and the formation of focal adhesions.
They also identified a number of known drug targets that were highly modulated in the tumor samples, including the protein Fyn, a target of the kinase inhibitor dasatinib (Sprycel); MAPK1, a target of drugs under development at Merck and Aeterna Zentaris and a downstream target of anti-HER and anti-MEK tyrosine kinase inhibitors; AKT1, a target of agents being developed by several pharma firms; and RAF1 and BRAF, both of which are targets of sorafenib (Nexavar). In total, the study identified as dysregulated 32 phosphopeptides targetable by 10 different agents.
Ultimately, Proteome Sciences hopes to use the Sysquant assay to help guide personalized therapies for various cancers, Ian Pike, the company's chief operating officer, told ProteoMonitor.
"The dysregulation of these signaling pathways is fundamental to disease and the response to treatment," he said. "So we need to know much more about the signaling processes in each individual to match the right drug to the right patient."
Such efforts exploring the potential of phosphoproteomic analyses to guide cancer therapy have been underway among proteomics researchers for several years, but the vast majority of this work has been done using antibody-based techniques like reverse phase protein arrays.
For instance, in perhaps the highest profile such study to date, researchers affiliated with breast cancer charity the Side-Out Foundation presented last June findings on the use of various omics data, including phosphoproteomic profiles developed via RPPA, in personalizing breast cancer treatment.
In 13 of the 25 subjects, these molecularly guided therapies extended progression-free survival by more than 30 percent compared to the patients' last treatment regimen.
RPPA has typically been used for this work due to the fact that the size of clinical samples available for such research is often too small for mass spec analysis. The PLOS One Sysquant study fits within a broader effort in the proteomics community to overcome this limitation and develop mass spec workflows suitable for the study of commonly available clinical samples like core needle biopsies.
In November, for instance, UCLA researchers used mass spec to analyze tyrosine kinase activation profiles in prostate cancer metastases. The month prior, UK-based proteomics company Activiomics announced plans to use its mass spec-based TIQUAS system for phosphoproteomic analysis of clinical samples, though it is unclear if it will continue that work in light of its recent acquisition by Retroscreen Virology Group.
In the case of the PLOS One work, Proteome Sciences was able to obtain the resected tumors through its collaborators and co-authors at King's College Hospital, which maintains a biobank of such specimens, Pike said.
He noted, though, that the firm was also exploring applying the technique to smaller core needle biopsy samples, with apparent success.
"We've done some studies [using core needle biopsy samples] in other therapeutic areas, and it looks like we need about a milligram of protein total [for the Sysquant assay], which means somewhere between 50 milligrams and 70 milligrams wet weight of tissue," Pike said, adding that this figure was "compatible with a core needle biopsy."
This is still significantly more sample, however, than is required by RPPA, which typically requires in the picogram to nanogram range of protein lysate. Another potential question is whether, in reality, a full core needle biopsy will typically be available for phosphoproteomic work given the variety of other analyses – including genomic and immunohistochemical – that clinicians will likely want to perform. Also a potential issue is whether enough sample will remain in the event that approaches like laser capture microdissection are used to reduce interferences due to tumor heterogeneity and stromal cell content.
Assuming the field is able to overcome mass spec's current sample requirement restrictions, though, approaches like Sysquant offer a potential advantage over RPPA in that mass spec is inherently more specific than antibody-based approaches and can measure thousands of proteins as opposed to the several hundred commonly measured on RPPA platforms, Pike said, suggesting that such a global view might further improve the personalized treatments thus far developed using antibody-based approaches.
He said that Proteome Sciences is currently planning trials similar to the Side-Out study in which it will use Sysquant data to tailor therapies to patients.
In addition to smaller sample requirements, faster turnaround will also be required if the assay is to become a true clinical tool. In the PLOS One study, the researchers were able to process and analyze 10 samples in roughly four weeks. Pike said they were now working to get this down to the one to two week range, largely by automating portions of their data analysis.
The PLOS One work was done on a Thermo Scientific LTQ Orbitrap Velos Pro, but the assay will also receive boosts in speed and sensitivity from the company's shift to Thermo Fisher Scientific's Orbitrap Fusion instrument, Pike said. He noted that they began using the Fusion for Sysquant work at the beginning of the year.
With the Fusion, the company expects to be able to perform the mass spec portion of the analysis on around 20 to 30 samples per week. The move to the new system has also increased the depth of their coverage, said Pike, noting that in recent work they are measuring around 18,000 different protein forms, including around 8,000 for which they can quantify both phosphorylated and unphosphorylated peptides.