The search for cancer biomarkers has proven to be a long, frustrating, and mainly fruitless exercise. Despite the substantial investment, no new markers suitable for large-scale screening protocols have achieved FDA approval. Yet, improvements in investigative tools, as well as innovative computer analysis, offer hope that the situation may be changing, and newly characterized candidates may prove to be clinically useful. At Visiongain’s recent “Biomarker Conference”, a number of presenters reported on putative biomarkers.
Diane Longo, Ph.D., a research scientist at Nodality, discussed the use of single-cell network profiling in the pathway analysis of cancer as a means to generate actionable clinical information. “This approach measures alterations in signaling pathways that drive disease processes and the response mechanisms of the host,” she explained.
The general protocol followed by Dr. Longo and her colleagues is to obtain blood or bone marrow samples from patients, expose the cells to stimulating or inhibitory factors, fix, permeabilize, and then stain the cells with flurophore-conjugated antibodies that recognize either extracellular surface markers or intracellular signaling molecules. The cells are then subjected to multiparameter flow cytometric analysis. This approach allows for the measurement of evoked signaling at the single-cell level, capturing the response of rare cell populations and revealing signaling heterogeneity.
Translating the biological characterization of patient samples into information that can be used to guide clinical decision making required an “industrialization” of the assay that could be run in a standardized high-throughput format to ensure that the results were highly reproducible.
The single-cell, network-profiling approach of mapping signaling networks has applications in disease characterization, clinical medicine, and drug development. As an example, Dr. Longo talked about her team’s work with acute myeloid leukemia (AML) in which they performed a functional characterization of intracellular signaling pathways in myeloblasts from patient samples. These studies revealed significant signaling heterogeneity among acute myeloid leukemia samples from genetically defined subgroups. Specifically, signaling heterogeneity was observed within subclasses defined by the presence or absence of a mutation in the FLT3 receptor kinase.
FLT3 internal tandem duplication mutations are among the most frequent mutations in AML and have been shown to negatively affect outcome in this disease. A functional characterization of intracellular signaling networks within individual cells from AML samples allowed for a subclassification of patients beyond their molecularly determined FLT3 mutation status and may have value in predicting clinical response.
“These studies allow us to conclude that we can identify and monitor rare cell populations,” Dr. Longo explained. “Moreover, the combination of different pathway information could provide assays to inform clinical decision making. Ongoing training and validation studies will critically test the clinical validity of single-cell network profiling for disease characterization and management in acute myeloid leukemia.”
Searching the Secretome
Daniel Chelsky, Ph.D., CSO at Caprion Proteomics, presented his company’s proteomics methodology to biomarker discovery and validation. “We combine a search for biomarkers in biological fluids with discovery in tissues, driven by highly multiplexed assays.”
Dr. Chelsky’s overall developmental strategy is based on MRM, a mass spectrometry assay that can specifically target large numbers of proteins. Promising biomarkers can be assembled by trolling the literature, proteomics assays, and transcript profiling. Through a validation funnel, the candidates are screened and winnowed down to the most promising possibilities.
“From this data, expression in the source tissue predicts expression in blood,” Dr. Chelsky said. “We can also determine the specificity of these proteins by building a ‘Body Atlas’ catalog of shared vs. specific secreted proteins from a wide range of tissues. We believe that this approach, combined with highly multiplexed multiple reaction monitoring, provides a powerful tool for biomarker verification.”
The company is focused on biomarker discovery using label-free, gel-free, quantitative mass spectrometry—a nonhypothesis-driven strategy, or “fishing expedition” in which thousands of proteins are profiled, narrowing down to dozens or hundreds of differentially expressed proteins. Subsequently, these are verified through a targeted approach involving multiplexed, quantitative multiple-reaction monitoring by mass spectrometry. This strategy allows large numbers of samples to be analyzed with a final aim of validation for regulatory compliance.
Caprion has pursued a number of malignant conditions such as lymphoma within the CNS. Diagnosis of this condition is fraught with difficulty, requiring expensive imaging or invasive biopsies that are dangerous and not always possible. As an alternative, cerebrospinal fluid from patients with lymphoma and matched controls was investigated, resulting in some 76 candidate markers. Among these, antithrombin III levels were found to be an accurate predictor of disease progression, providing better sensitivity and specificity than the conventional cytology, according to the company.
Given that secreted proteins are perhaps a thousand times more concentrated in the Golgi apparatus than in the blood, targeting this subcellular organelle and the attendant secretory vesicles to reveal the secretome is another promising strategy to detect markers present in very low concentrations. Caprion staff investigated the presence of biomarker proteins in prostate tumors and their normal counterpart.
Among 60 differentially expressed proteins, they were able to identify seven that were previously identified by other investigators in plasma. Two of these, prostate specific antigen and macrophage migration inhibitory factor, were measured by ELISA in the plasma and were strongly correlated with measurements in the patients’ tumors.
Another series of investigations identified secreted protein indicative of progressive type 2 diabetes. By focusing on those secreted proteins found only in the beta cells, Caprion researchers identified almost 300 proteins specific to the beta cells that are currently being evaluated as candidate biomarkers.