NEW YORK (GenomeWeb) – Drug and diagnostics giant Roche and proteomics firm Biognosys have completed a study indicating that data-independent acquisition (DIA) mass spec is suitable for use in clinical trials.
The companies presented data at the Mass Spectrometry Applications to the Clinical Lab (MSACL) annual conference this month from a mock clinical trial in which Roche used DIA mass spec to collect proteomic data on 30 colorectal cancer specimens.
In the study the researchers used four-hour LC gradient and mass spec analysis on a Thermo Fisher Scientific Q Exactive HF instrument to identify roughly 9,000 proteins across the 30 samples and with coefficients of variation below 10 percent, conducting the assays in compliance with GCP guidelines and on a timeline suitable for clinical trial work.
Axel Ducret, a principal scientist at Roche and one of the leaders of the effort, said he believed DIA mass spec could allow pharma firms to collect proteomic information on clinical trial participants in much the same way RNA-seq has enabled the collection of transcriptomic data. Such data could, for instance, help companies better understand the mechanisms and on- and off-target effects of their drugs and identify markers and molecular profiles linked to patient response, he said.
"I'm very much convinced that [proteomic information] is complimentary to what RNA-seq or genomic information can tell you," Ducret said.
He cited the example of genetic mutations, noting that while they are of great interest in drug development, often "the downstream effects of those mutations are not very well understood. And I think that having the protein-level data is going to be very important to better understanding what is going on."
The notion that proteomic data can bolster genomic and transcriptomic analyses is widespread, particularly in proteomic circles, and has led to growing interest in proteogenomic experiments. Proteomic techniques have traditionally not offered depth of coverage or throughput comparable to genomic and transcriptomic analyses, however.
DIA mass spec has changed that, Ducret said, noting that the technique offers highly reproducible label-free quantitation that can be run with relative high throughput and a depth of coverage that is roughly equivalent to that offered by RNA-seq. Another key, he added, is the fact that the DIA assays were compatible with formalin-fixed paraffin-embedded (FFPE) samples, which are commonly used in clinical trials.
"So, it is possible to run a proteomics study using the same material and roughly the same amount of tissue that is used for RNA-seq… and both approaches can be run in parallel," he said. "You can do a proper proteomic analysis that can be matched to an RNA-seq analysis and get a comprehensive dataset that you can use to much more powerfully model what is going on."
He noted that Roche has used DIA for preclinical work for several years, including in collaboration with Biognosys.
DIA mass spec has become widely used in proteomics research over the last five years, but the technique has not been used in the sort of GCP-compliant workflows necessary for clinical trial work, Ducret said.
The technique is attractive due to its ability to provide highly reproducible quantitation across large numbers of samples. Unlike conventional shotgun mass spec, DIA mass spec selects broad m/z windows and fragments all precursors in that window, which allows the instrument to collect MS/MS spectra on all the ions in a sample. This means the instrument looks at the same peptides in every sample, avoiding the stochastic sampling problem that is an issue in conventional proteomic workflows, making for more reproducible quantitation.
Isobaric labeling approaches like TMT are also commonly used for quantitative proteomics work, but these methods can be difficult to apply to large numbers of samples. Currently, a single TMT experiment can analyze a maximum of 10 samples, and while researchers can use reference samples to compare results across different TMT experiments, this approach can be cumbersome.
The colorectal cancer project provides a proof-of-concept that DIA is compatible with such workflows, and Ducret said he believed Roche would have GCP-compliant DIA workflows up and running within a short period of time — perhaps a few months or so.
For Biognosys, development of a GCP-compliant DIA workflow will allow it to move its mass spec services into a new stage of the drug development process. The Schlieren, Switzerland-based company offers a variety of mass spec services for pharma and other customers but has focused much of its work in recent years on DIA-based approaches.
Biognosys CEO Oliver Rinner said the company expects that it will see demand for GCP-compliant DIA analyses among pharma customers that have to date been using its DIA services in the research setting. Like Ducret, Rinner suggested companies are interested in using proteomic data to add power to their RNA-seq analyses, noting that drugmakers are still working to understand how to effectively utilize omics technologies.
"It is obvious that [omics datasets] contain a lot of information that could help you to, for instance, stratify patients in a clinical trial," he said. "But I think that for all these technologies it remains to be shown that they can really impact the efficiency of a clinical trial. It's clear that this is information that [drugmakers] need, but it is still a challenge for everyone in the field to make sense out of this data."
Ducret said that he believes Roche is at the forefront among pharma firms in moving towards implementing DIA within clinical drug trials, but that it is not an outlier.
"If you speak to other people in the field, many are also very interested in this approach," he said, adding that the colorectal cancer data he and his colleagues presented at MSACL provide evidence that such an approach can work.
"I think now we have a very good case that shows that if we need to do some exploratory biomarker work using DIA, that it works and that it can be done in a given timeframe," he said.
In additional to the DIA work done by Biognosys, the project used selected reaction monitoring (SRM) mass spec assays to 12 potential colorectal cancer protein markers developed by Montreal-based CRO Caprion Biosciences.
Ducret said that while this work was not as novel as the DIA portion of the study, it was nonetheless significant in that it demonstrated that targeted proteomic assays could be developed according to GCP guidelines and quality criteria from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) and on a timeline compatible with a clinical trial.
Caprion already had a GCP SRM workflow in place and has validated several multiplex SRM assays for regulatory submissions, said Lorella Di Donato, the company's vice president, scientific operations. However, she noted that developing the 12-marker panel was not without its challenges.
In particular, Di Donato stressed the need to carefully select appropriate peptides as surrogates for the target protein and to correctly interpret how the signal of the different peptides corresponds to expression at the protein level.
She noted that she and her colleagues used the peptide library developed by CPTAC to streamline the selection of the best peptide surrogates and synthesize standards based on them and suggested that this resource would help meet the "demand to set up biomarker assays in a relative short timeframe due to timeline constraints often present in clinical trials."