Cancer Biomarker

Investigators:  Sahar Sibani, Ph.D., Alex Mendoza (technician), Eugenie Hainsworth (engineer),  Ralph Eugene (technician)

Collaborators: Karen Anderson (DFCI), Marty Sanda (BIDMC), Dan Cramer (BWH), Xihong Lin (HSPH), Andrew Godwin and Paul Engstrom (Fox Chase), Jeffrey Marks (Duke)

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. This points to a need to identify new autoantigens that would enable multiplexed testing for cancer. Early studies have shown that using a panel of autoantibodies improves both sensitivity and specificity of detection. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring.

We have invented a novel form of protein microarray, called Nucleic Acid-Programmable Protein Array (NAPPA), which replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA (Figure 1). Our technology exploits the ability to transfer our ORFs into specialized tagged expression vectors, the new expression clones are then spotted on the array, the proteins are then produced in situ in a cell-free system and immobilized in place upon their synthesis. This minimizes direct manipulation of the proteins and produces them just-in-time for the experiment, avoiding problems with protein purification and stability (Science. 2004 305:86). A next generation method for these arrays has been developed that allows thousands of proteins to be produced simultaneously in situ, and with remarkably consistent protein levels displayed (Nat Methods. 2008 5:535).

The overall goal of this project is to identify autoantibody biomarkers in sera that can be readily used for the early detection of cancers. We are producing protein microarrays using our method and screening this against sera from both cancer patients and healthy controls, in order to find autoantibodies that are specific in patients (Figure 2). We have first focused these experiments on breast cancer, and are expanding to other diseases, including ovarian cancer (Dan Cramer), prostate cancer (Marty Sanda), and lung cancer (Proteomika).

Figure 1. - See full version

The NAPPA method.

(A) DNA is printed on the array and converted into protein.

(B) The range of protein concentration (y-axis) is limited to single logarithm. 93% of proteins are within two fold of the mean.

(C) There are no apparent biases for protein classes on NAPPA.

Figure 2. - See full version

Using protein microarrays to screen serum for immune responses.

(a) Identifying immunodominant antigens in patients infected with pseudomonas,

(b) tracking the immune response in mice vaccinated with an attenuated bacterial strain,

(c) tumor antigens in prostate cancer and (d) autoantibodies in type I diabetes.