“Just knowing whether a protein is present usually is not the most important information,†said Steven M. Kornblau, MD, professor in the department of leukemia and stem cell transplantation at the MD Anderson Cancer Center in Houston. “It is important to understand if a protein has undergone a post-translational modification, such as phosphorylation, which typically converts the protein to its active form, for example. You can’t get that kind of information from messenger RNA profiling.â€
Dr. Kornblau’s lab uses proteomic tools to characterize hematologic malignancies. For proteomics researchers, protein-level information is the ultimate goal in cancer research because the protein function regulates the phenotype of wild-type cells and cancer cells – providing information about a cell’s response to its environment and its pathogenetic state.
“If there is a mutation in the genome, it is good to know, but it is better to know if that mutation is transcribed into RNA and then, third, if the RNA is translated into an active protein,†he explained. “These ‘third protein level’ data – the proteomic data – are ultimately better positioned than genomic or transcriptome data to tell us how much of an actual effector there is in the activated state.â€
The Proteomics Toolkit
Most techniques used in proteomic analyses require that protein samples are first fractionated to isolate various subcellular organelles. Then samples are separated and analyzed using mass spectrometry.
Proteins can be separated using affinity separation methods, by two-dimensional gel electrophoresis, or one- or two-dimensional chromatographic separation. Two-dimensional gel electrophoresis is frequently used to separate complex mixes of proteins by two protein properties, isoelectric point and molecular weight, to study both qualitative and quantitative protein changes among different samples. One-dimensional liquid chromatography, when used with electrospray mass spectrometry analysis, generates high-quality data about the types of proteins in a sample, but this tool cannot be used to quantitate proteins.
The next step after protein separation is protein content analysis, including identifying even small amounts of proteins, using mass spectrometry. Mass spectrometry is used in hematology biomedical research because the tool does not require a large sample to run the analysis and can be used to analyze proteins, peptides, and their post-translational modifications.
“We can identify minor modifications between two proteins or two peptides, including oxidation of amino acids and phosphorylation,†explained Dr. Tissot. “This is a fantastic tool to characterize proteins and their post-translational modifications.â€
Another protein analytical tool is reverse phase protein array (RPPA), a sensitive and high-throughput technology to quantitate hundreds of proteins and phosphoproteins in biological or clinical samples. In this type of analysis, protein lysates from as many as 1,000 samples are printed onto a slide and probed with the same antibody.
The technique allows for reproducibility and sensitivity but does not allow for the assessment of an entire proteome. “This is a great technique for discovering targets and biomarkers,†said Dr. Kornblau, “but it is too time-consuming to have potential in the clinic.â€
Characterizing Diseases
Proteomics is helping researchers better understand hematologic malignancies and other blood disorders, such as genetic coagulation disorders and amyloidosis.
“It is important to use proteomics for hematologic diseases where there is accumulation of proteins, as is the case with amyloidosis,†said Dr. Tissot. “It is possible to take a patient’s tissue sample and do direct proteomic analysis to characterize the accumulation of these proteins. Now, we also can isolate single cells for proteomic analyses, identifying different malignant cell populations or different types of cells that are found during differentiation and maturation of white blood cells.â€
In 2005, scientists at the National Cancer Center Research Institute in Japan conducted a large-scale proteomic analysis of cell lines derived from human lymphoid cancers, including Hodgkin lymphoma and Burkitt’s lymphoma, to identify unique protein patterns among these B- and T-cell malignancies.7 They found that Hodgkin lymphoma has a unique protein expression profile that differentiated it from wild-type B and T cells and other blood cancers; ultimately, they hope to use these patterns as novel diagnostic markers for the disease.
Similarly, in 2009, Dr. Kornblau and his colleagues used RPPA to classify acute myeloid leukemia (AML). They assayed leukemia-enriched cells from 256 patients with newly diagnosed AML, identifying a total of 51 proteins that contribute to apoptosis, cell cycle regulation, and other functions often mutated in cancers. These proteins provided prognostic information that was distinct from the cytogenetics of AML cells and correlated with remissions, relapse, and patient survival.8 His team also discovered that they could obtain the same protein levels whether the samples were derived from a blood draw or bone marrow.
More recently, in 2019, they were able to probe their AML samples using antibodies that targeted many more proteins – more than 250 of them.9
In another project, Dr. Kornblau, along with pediatric oncologist Terzah Marie Horton, MD, PhD, from Baylor College of Medicine and Texas Children’s Hospital, led a team that analyzed the proteomic landscape of pediatric AML to identify potential new drug targets. The researchers found distinct protein signatures that, when combined with the cytogenetics of a patient’s AML, could add to the prognosis information for patients.10
They also assayed cells for the presence of a select group of proteins in pediatric patients with AML and identified select patients that could benefit from treatment with the proteasome inhibitor bortezomib in combination with a standard chemotherapy regimen.11
“We found protein signatures from patients that correlated with a benefit from the addition of bortezomib, as well as those groups that appeared to have no benefit from the addition of bortezomib,†Dr. Horton noted.
Drs. Horton and Kornblau said that, after additional proteomic analyses, they plan to conduct a study in pediatric AML in which patients will be screened for these protein signatures and given bortezomib if their signature predicts a response. “This research is a ‘poster child’ example of where proteomics is allowing us to identify a subgroup of patients that are likely to benefit from a drug,†added Dr. Kornblau.
All these disease characterizations have yet not made it into general medical practice, according to Drs. Tissot and Kornblau. So far, identifying a single or a few protein biomarkers of disease has not proven to be a reliable way to make an early diagnosis of a hematologic malignancy, Dr. Tissot noted.
He sees the greatest translational potential for bringing proteomic data into the clinic in developing novel immunotherapies. “It is important to identify novel target peptides that are produced by cancer cells that we can then use for immunotherapy such as chimeric antigen receptor T-cell therapy and vaccination,†he said.
The University of Lausanne, where Dr. Tissot conducts research, is now working on a large-scale analysis of peptides that are uniquely produced by tumor cells and that can be recognized by T cells to develop new immunotherapy targets. “This is a huge effort and proteomic approaches are essential because we need to identify – accurately – the actual peptides and proteins that are being made by the cancer cells,†he noted.
Limitations and Complementation
One drawback of proteomic analysis is the time it takes to go from a sample to a full analytical result, which is typically on the order of several weeks for a large-scale proteome data set. Single cell sorting followed by mass spectrometry is quicker, by about one day.
Proper protein sample preparation is crucial, according to Dr. Tissot, as proteins are far less stable than DNA or even RNA. Samples need to be processed quickly to avoid delays between blood collection and isolating plasma or cell types that may allow time for artifacts to be introduced.
Another limitation is that, so far, it is difficult to accurately quantitate protein levels in a sample using available mass spectrometry techniques.
For Dr. Tissot, combining proteomic approaches with other modalities, including analysis of the genome and the RNA in cancer cells, is critical “to have a global view of what happens in cancer cells and the differences in their metabolism.â€
“This is basic research that is crucial to understand how these cells function and respond to their environment,†he added.
“Proteomics is not yet daily lab work for hematologists,†said Dr. Tissot. “These are not yet widespread approaches, although they are coming for blood cancer, immunotherapy, and protein-based diseases such as amyloidosis. It’s important that we develop these protein tools.†—By Anna Azvolinsky