Autoantibody Biomarkers in Ovarian CancerMonica Muthaiya

Department of Pharmacology, Rush University

Faculty Advisor: Dr. Judith Luborsky

Background

Cancer is defined as a malignant growth or tumor caused by abnormal

and uncontrolled cell division that may profuse to other parts of the body

through the lymphatic system or the blood stream. Ovarian cancer is a

type of cancer that forms in tissues of the ovary, in which the ova are

formed (5). Ovarian cancer occurs when ovarian cells develop in an

unrestrained & unnatural manner (2). Ovarian cancer is most often seen

in women between the ages of 40 to 60 and rarely in adolescents.

The survival rate for ovarian cancer is low. Most women are diagnosed at

late stages after tumor metastasis and less than 20% survive 5 years.

Early detection methods are clearly needed. A substance that could be

measured in blood would be efficient, but most tumor proteins have not

proven useful. However, patients have a characteristic immune

response to cancer, that can be measured in blood as auto antibodies.

Information on the behavior of specific antibodies and their ability to

predict ovarian cancer is needed for antibodies to be used clinically.

Studies over time are difficult in humans (3).

Hens are the only known spontaneous model of ovarian cancer that

develop the cancer at a high rate. Using hens allows researchers to

identify processes involved in spontaneous tumor formation and

progression, and to identify possible biomarkers that predict the tumors.

Dr. Luborsky’s laboratory showed for the first time that SELENBP1 is an

auto antigen in ovarian failure associated with an autoimmune disease in

young women. (1) They also showed that SELENBP1 is expressed in

both normal ovaries and ovarian tumors in the hen. (4) Similar to

humans, hens with ovarian tumors make auto antibodies to SELENBP1

making it possible to examine the antibody response as hens develop

tumors. The antibody titer is a measurement of how much antibody an

organism has produced, expressed as the greatest dilution that still gives

a positive result. ELISA is a common procedure in determining antibody

titers.

Purpose & HypothesisPurpose: The aim was to determine if the titer of SELENBP1

autoantibody changes as the hens develop tumors.

Hypothesis: If ovarian cancer develops in a hen, then the titer

will decrease during the progression from normal ovary to tumor

occurrence.

VariablesIndependent Variables: Hen with Ovarian Cancer or No Cancer

Dependent Variables: SELENBP1 Antibody Level and Titer

Procedure1. Animals: White Leghorn laying hens (2.5 - 3 years old) were

maintained at the Poultry Research Farm of the University of

Illinois at Urbana-Champaign, under a controlled light regimen

(14 h light:10 h dark) with food and water provided ad libitum.

2. Hen Selection: Based on ultrasound morphology , hens with

normal ovaries were selected.

3. Blood Collection: Serum was serially collected at ~2-4 month

intervals. Blood was obtained from each hen’s wing vein and

serum stored at -80°C.

4. Experiment Completion: At the completion of the study (6-14

months) hens were euthanized according to the approved

Institutional Animal Care and Use Committee (IACUC) protocol.

Ovarian tissue and blood was collected. The presence of a

tumor was determined by gross morphology and histology as

described previously (ref) .

5. Immunoassay to detect autoantibodies: Conduct a standard

immunoassay to measure autoantibody against SBP1. Use

purified recombinant SBP1 as an antigen to capture the serum

antibody, and anti chicken IGY_HRP (horseradish peroxidase)

as the secondary antibody. to detect bound serum antibodies.

Read the optical density using a Spectrophotometer with

wavelength set to a 450nm and 580nm as a reference

wavelength.

6. Safety procedures: While conducting this experiment, all

safety protocols were followed. All materials were handled with

gloves while wearing a lab coat.

Data

* As the dilution increases, autoantibody signal decreases, like

expected. By testing different dilutions, you can reduce error in your

experimental design. When collecting data for different dilutions, I

observed the fading of the signal.

* In the Normal samples, the regularity between the dilutions

remained as expected, solely with DOD63 & DOD33 being exceptions.

DOD63 had a 9 month difference from the first scan to the final scan.

The dates were not recorded when collecting the data, so the data

from this hen is not very reliable. In the OVCA samples, the samples

were very consistent, significantly fading out on all but sample DOD62,

suggesting that errors may have occurred and the data from this hen

may not be reliable either. DOD62 showed abnormalities within

dilutions, suggesting that errors may have occurred: while creating the

dilution, in the ELISA wells being tested, when extracting a sample

from the hen, or within the hen itself. DOD62 was diagnosed with

stage 3 mucinous ovca, a cancer where the mucinous tumors are filled

with a mucous-like substance. This type of OVCA is unpredictable and

may have led to the increase in signal, as dilution increases. From the

data above, OVCA samples contained very minimal sources of error

within dilution variations.

* The data suggests that there is no significant difference in

percent change in OD value from hens with OVCA and the control

group. The normal hens have great fluctuation and higher signals than

the OVCA group. Therefore, normal hens have a lower titer in

comparison to the OVCA. The titer of SELENBP1 autoantibody

remains constant as the hens develop tumors.

Conclusion

The aim was to determine if the titer of SELENBP1

autoantibody changes as the hens develop

tumors. Based solely off the data extracted in this

experiment, there is no significant difference in

percent change OD values between the hens with

OVCA and the control group. However, various

papers written by the Department of Pharmacology

at RUSH University suggest that SELENBP1

autoantibody may be a potential biomarker for

Ovarian Cancer.

The original hypothesis stated that “If ovarian

cancer develops in a hen, then the titer will

decrease during the progression from normal

ovary to tumor occurrence.” The percent change in

OD Value remained fairly close to 1. The

hypothesis is mainly refuted, as the titer didn’t

significantly increase or decrease. Further

experiments will need to be executed to come to a

decisive conclusion.

References1. Edassery, S. L., Shatavi, S. V., Kunkel, J. P., Hauer, C., Brucker, C., Penumatsa, K., Yu, Y., Dias, J. A., & Luborsky, J. L. (2010). Autoantigens in ovarian autoimmunity associated with unexplained infertility and premature

ovarian failure. Fertility and Sterility, 94(7), 2636–2641. Retrieved from http://www.sciencedirect.com/science/article/pii/S0015028210006096

2. Kim, A., Ueda, Y., Naka, T., & Enomoto, T. (2012). Therapeutic strategies in epithelial ovarian cancer. Experimental and Clinical Cancer Research, 31. Retrieved from http://www.jeccr.com/content/31/1/14

3. Slotman , B. J. (1988). Ovarian cancer (review). Etiology, diagnosis, prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer Research. Retrieved from

http://europepmc.org/abstract/MED/3291746/reload=0;jsessionid=bxXI0bWGzq7oiufhjjE5.36

4. Stammer, K., Edassery, S. L., Barua, A., Bitterman, P., Bahr, J. M., Hales, D. B., & Luborsky, J. L. (2008). Selenium-Binding Protein 1 expression in ovaries and ovarian tumors in the laying hen, a spontaneous model of

human ovarian cancer. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18272210

5. Tan , E. M., & Zhang, J. (2008). Autoantibodies to tumor-associated antigens: reporters from the immune system. Immunological Reviews, 222(1), 328-340. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18364012

The Effect of

Ovarian Tumor

Development

on Autoantibody

Titer

The Effect of

Ovarian Tumor

Development

on Autoantibody

Titer

Figure 1. Expression of SELENBP1 in normal hens

ovaries. Each row contains an H&E stained section

(A & D) and a corresponding example of the

SELENBP1 expression (B, C, E & F) at two

magnifications (20X and 120X). SELENBP1 is

predominantly found in surface epithelium cells

(arrows) of normal ovaries.

Acknowledgements:

I would like to extend my thanks to Dr. Judith Luborsky, Seby Edassery, Yi Yu and the faculty at the Department of Pharmacology: they gave me great advice on future plans for research, medicine, and experiments.

Figure 2: The figure on the left shows the

final data collected from this experiment. It

shows the percent change in OD value from

the first to last scan between OVCA and

normal hens within each dilution.

a | A shows an example of a high-grade

carcinoma, morphologically consistent with

the serous subtype, with a small follicle

towards the bottom left-hand corner.

b | B is an example of a low-grade

endometrioid carcinoma showing simple

glandular structures in a back- to-back

arrangement with little stroma intermixed

between the glands (scale bar = 100 μm).