The Use of Soluble, Salivary c-erbB-2 for the Detection and Post-Operative Follow-up of Breast Cancer in Women: The Results of a Five-year Translational Research Study

C. Streckfus and L. Bigler

The goal of this study is to identify and isolate salivary biomarkers that can definitively show the presence of breast cancer. An initial study by these investigators was first conducted to answer the question of whether salivary biomarkers specific to breast cancer could indeed be found. This study compared the saliva compositions of three groups of women: healthy women, women with benign breast tumors, and women with malignant breast tumors. This study showed the protein product of the oncogene c-erbB-2 and CA 15-3 as significantly higher in the malignant cancer case as opposed to the healthy and benign groups. This identification was conducted by the simple use of an ELISA kit. Based on these results, the group proceeded to choose c-erbB-2 for further investigation.

Relative Concentration Differences Between Healthy, Benign, and Malignant Groups. There was no significant difference in c-erbB-2 concentration between healthy and benign, but malignancy was significantly different.

This investigation evaluated c-erbB-2 as a marker, as well as elucidating the signaling pathway that gave rise to this high c-erbB-2 concentration. The high concentration of c-erbB-2 in saliva was consistent with the patient’s high concentration of c-erbB-2 in serum. C-erbB-2 was determined to be a superior marker in detection of breast cancer in patients than CA 15-3 (which was used as a ‘gold standard’ in these experiments. Researchers also evaluated the possibility of alternate factors that could explain these high concentrations besides breast carcinoma. These included the patient’s age, weight, race, prescription medications used, and other illnesses. C-erbB-2 concentration in saliva was found to be independent of all these factors.

A third investigation was undertaken to identify whether c-erbB-2 was a good temporal indicator, i.e. before, during and after therapy. In order to do this, further clarification on the c-erbB-2 pathway from start to presence in saliva needed to be determined. Western blot techniques provided several answers. It was determined that the submandibular salivary gland contributed the most to c-erbB-2 salivary concentration. Also, due to the thicker bands from saliva samples, saliva was determined to have a higher baseline concentration than serum. SELDI techniques (Surface-enhanced Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) were used to confirm the ELISA results.

Western Blots of Saliva and Serum samples taken from Malignant patients. The darker bands on the saliva show higher concentration of c-erbB-2 in the saliva when compared to serum

These investigations gave insight to the overarching question: How did c-erbB-2 enter the saliva? The paper postulated that the protein was not transported through any conventional means such as convection or diffusion, due to its large size. Rather, they postulated that over-expression of c-erbB-2 caused a humoral response in the salivary glands and thus the concentration was increased locally.

This investigation in protein tracking and use of ELISA is an important step forward in finding a diagnostic tool for breast cancer. ELISA is a relatively inexpensive technology, essentially a 9 x 12 microtitier plate possessing a monoclonal antibody for the antigen of interest. Since anti-X can be created from any species (mouse, rabbit, pig) it is relatively inexpensive. ELISA assays can be conducted in a matter of a few hours, making the process very simple. Also, the tests are relatively specific; differences in wavelength absorbances are translated to a scale of concentration. Since c-erbB-2 is significantly overexpressed in malignant cancer patients, ELISA is sufficiently specific to detect significant changes in concentration.

The fact that ELISA enables diagnosis through saliva is noteworthy. Saliva is easily collected and non-invasive. The ease of taking this test would encourage women to test themselves more frequently, and thus enable early detection of malignancy. Also, women with benign tumors could use ELISA as a means for finding out whether their cancer is malignant or not without getting a biopsy.

NOTE: While this is a promising technique, it must be mentioned that c-erb-2/HER2 is overexpressed in only 20-30% of breast cancer cases. Thus, this technology would have limited application, similar to the nanoshell detection case.