One barrier to analyzing proteomic response is the large amount of specimen needed for measurements at each time point. Researchers are now using nanoscale methods to define biologic signatures and measure proteomic response to targeted therapies in hematologic and solid tumors, as reported study at the Molecular Diagnostics in Cancer Therapeutic Development conference.
Alice C. Fan, MD, a medical oncologist at Stanford University, described a highly sensitive nano-immunoassay that quantifies unphosphorylated single and multiple phosphorylated isoforms of proteins in specimens from minimally invasive blood draws or fine-needle aspirates.
The assay, performed on a microfluidic Nanopro1000 from Cell Biosciences, can produce reliable nanoimmunoassay results with only four nanoliters of lysate. Dr. Fan said that after studying the nano-immunoassay in several clinical trials, it was shown that protein profiles in the RAS and MAP kinase pathways could distinguish tumor cells from normal cells.
To investigate the diagnostic potential, her team collected some 300 specimens from normal controls and patients with hematopoietic and solid tumors, finding that phosphoprotein profiles in tumor cells were able to distinguish tumor from normal cells. Further, patient tumors could be grouped based on different patterns of the percentage ERK and MEK phosphorylation.
In one prospective study of a novel cell cycle inhibitor in MDS, the nano-immunoassay was used to measure changes in MAPK and cell cycle proteins in leukocytes sampled during the study of a PLK1 cell cycle inhibitor. “We take cells from patients at each time point that they come in for the clinical trial visit, and we can measure transient decreases in certain molecules,” she said. “This again suggests that being able to resolve differences between unphosphorylated isoforms and single phosphorylations and double phosphorylations might provide useful information in trying to understand how the drugs actually work.”
In another prospective study, Dr. Fan tested atorvastatin in non-Hodgkin's lymphoma patients “to measure if the statin was actually doing what we predicted it to do in the preclinical studies.” After collecting blood samples before treatment on Day 1 and again on Day 8, she used the nano-immunoassay and multicolor intracellular flow cytometry to distinguish three patterns of signaling changes in tumor cells, monocytes, and T cells.
“By Day 8 we can measure significant differences in patients receiving atorvastatin,” she said. “The atorvastatin effect is differentially affecting the tumor cells and not the host immune cells. In fact, it indicates that the host immune cells are revved up when you treat with atorvastatin, which may have something to do with the immunomodulatory effects and how the tumor might actually be shrinking.”
—Robert H. Carlson