Cancers are frequently associated with the abnormal expression and phosphorylation of oncogenes, and the ability to detect specific oncoproteins and their activated forms is a major goal in efforts to develop new treatments and methods for assessing response to treatments. Current methods for protein detection are relatively insensitive to small changes in oncoprotein activation underlying cancer signaling processes, and serial tumor sampling for assessing response to treatments is often precluded by the need for large numbers of cells for analysis. Alice C. Fan, MD, and Dean W. Felsher, MD, PhD, at Stanford School of Medicine, recently developed a nanofluidic proteomic immunoassay (NIA) that is capable of quantifying total and low-abundance protein isoforms in nanoliter volumes, allowing collection of samples through minimally invasive blood draws or fine-needle aspirates.

“NIA is an exciting new technology for rapidly measuring proteins in minimal amounts of clinical materials,” Dr. Felsher, Associate Professor, Medicine-Oncology, at Stanford, told The ASCO Post. “The method has been automated to incorporate a charge-based separation of proteins with a microfluidic platform and antibody-based detection. To date, we have used this method for nanoscale analysis of blood, bone marrow, and fine-needle aspirates. Eventually, this approach will also enable the direct assessment of targeted treatments that will assist in personalized cancer therapy.”

Technique

The NIA test developed and used in studies by Drs. Fan and Felsher (NanoPro 1000 Instrument, Cell Biosciences™) is capable of using samples as small as 25 cells to generate information on the post-translational status of signaling proteins. NIA uses a standard 384-well microplate and can analyze up to 96 samples with each run.

As with the Western blot technique, proteins in samples are separated, immobilized, and probed with antibodies. However, the NIA system uses capillary isoelectric focusing separation to resolve phosphorylation states of signaling proteins, with the separated proteins being immobilized to the capillary wall and probed with primary and secondary antibodies. The secondary antibody is HRP-labeled, permitting ultrasensitive chemoluminescence detection of proteins present in even low quantities.

Findings

The NIA has been shown to detect oncoprotein expression and oncoprotein phosphorylation in clinical specimens. Initial studies of specimens from normal controls and patients with solid and hematopoietic tumors showed that the immunoassay can detect phosphoprotein profiles in tumor cells that distinguish tumor from normal cells. Moreover, investigators determined that patient tumors could be grouped based on different patterns of percent ERK and MEK phosphorylation.

In particular, the NIA has been shown to be able to quantify amounts of MYC oncoprotein and B-cell lymphoma protein 2 in Burkitt’s and follicular lymphomas and identify changes in activation of ERK1 and ERK2, MEK, STAT3 and STAT5, c-JNK, and caspase 3 in imatinib (Gleevec)-treated chronic myelogenous leukemia cells. The immunoassay also has been able to measure an otherwise unanticipated change in the phosphorylation of an ERK2 isoform in patients with chronic myelogenous leukemia responding to imatinib and detect a decrease in STAT3 and STAT5 phosphorylation in patients with lymphoma treated with atorvastatin (Lipitor).

Clinical Trials

In the clinical trial setting, NIA has been used alone and in conjunction with multicolor flow cytometry (fluorescence-activated cell sorter, or FACS) to monitor changes in phosphorylation profiles among patients receiving novel agents for hematopoietic cancers. For example, NIA has been used to measure changes in MAPK and cell-cycle proteins in serially sampled leukocytes during a study of a new cell-cycle inhibitor in patients with myelodysplastic syndrome.

NIA was highly complementary to FACS for the detection of signaling changes in tumor cells, monocytes, and T cells in a trial of atorvastatin in non-Hodgkin lymphoma patients.  Pathways initially activated in tumor cells showed reduced activation with atorvastatin treatment, with reductions in phospho-STAT3 and phospho-STAT5 of up to 70% being observed, whereas pathways that were initially suppressed in tumor cells (including phospho-phospholipase Cγ) were normalized during treatment. Moreover, expression levels of proteins in apoptotic pathways (including p38) increased with treatment, and changes in signaling associated with atorvastatin treatment were found to be cell-specific, with effects on tumor cells being distinct from effects on nontumor T cells and monocytes.

Conclusions

These findings show that nanoscale proteomic technology can be used to identify and quantify changes in cell signaling and identify responses to therapeutic intervention in different cell populations using small clinical specimens that can be collected serially from patients with minimal invasiveness. These results suggest that this technology may allow identification of previously unidentified changes in signaling patterns in tumor cells that may prove important in development of targeted therapies or that may serve as response markers. Overall, nanoscale analysis appears to be a promising approach to identifying and measuring diagnostic markers of disease and assessing and monitoring markers of therapeutic response.

“NIA is an example of how new technologies might help us develop better ways to detect tumors earlier and make decisions more quickly,” commented Dr. Fan, Instructor, Medicine-Oncology, at Stanford. “It’s amazing that I can take a fine-needle aspirate from a patient, and within a few hours, know the levels of tumor signaling proteins in their cells. Developing the use of new technologies is an exciting collaborative effort among physicians, bench scientists, and our patients. Together, our goal is to complete clinical trials that show these analyses can help us optimize new treatments.” ■

Financial Disclosure: Dr. Fan reported no potential conflicts of interest. Dr. Felsher has previously served on the science advisory board for Cell Biosciences.

Resources

Cell Biosciences™ NanoPro 1000 system. Available at http://www.cellbiosciences.com/nanopro-1000.html.

Fan AC, Deb-Basu D, Orban MW, et al: Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nature Med 15:566-571, 2009.

Fan AC, Dermody J, Kong C, et al: Nano-immunoassay profiling of ERK and MEK isoforms in fine-needle aspirates of solid tumors. J Clin Oncol 28(15s):Abstract 2564, 2010.

Fan AC, Dermody JL, Kong C, et al: Nanoscale approaches to define biologic signatures and measure proteomic response to targeted therapies in hematologic and solid tumors. Presented at the Fourth American Association for Cancer Research International Conference on Molecular Diagnostics in Cancer Therapeutic Development, 2010. Abstract PR6. Available at http://www.aacr.org/Uploads/DocumentRepository/2010_conf/MD/moldia10_abstracts_proffered.pdf.

Fan AC, Orban MW, Shirer AE, et al: Nanoscale analysis of changes in signaling proteins in patients treated with single agent atorvastatin for low-grade or refractory NHL. J Clin Oncol 27(suppl):Abstract 11011, 2009.

Seetharam M, Tran M, Fan AC, et al: Treatment of higher risk myelodysplastic syndrome patients unresponsive to hypomethylating agents with ON 01910.Na. Blood 116(21):Abstract 4010, 2010.