Pap Test Project

Each year more than 21,000 women in the U.S. are diagnosed with ovarian cancer, primarily with late stages of the disease. Consequently, the five-year survival rate is only 45%. Early detection of ovarian cancer is the key to better survival rates, yet a screening tool has yet to be developed that is adequately sensitive and specific to the general population.

For decades, researchers have been using biospecimens (e.g. blood and tissues) taken from women with late stages of ovarian cancer in an attempt to discover biomarkers that could be used to detect early stages of ovarian cancer. 

The OCEDP's solution – the Pap test.
Screening for cervical cancer by Pap tests has been routinely performed for over 50 years. The liquid-based Pap test has an easy collection method whereby cervical cells are placed into an alcohol-based fixative and then examined for abnormal cells occasionally, ovarian cancer cells have been identified in Pap tests. 

A major focus of the OCEDP is to discover and verify ovarian cancer biomarkers in Pap test fixative samples from patients at early stages of the disease. At the University of Minnesota, the cytopathologists see over 60,000 Pap tests annually. In our Gynecologic Cancer Clinic, we have been performing "Mock Pap tests" for several years, and have accrued hundreds of fixatives and swabs from women with ovarian cancer and other gynecological conditions, as well as healthy women. The OCEDP will be the first to analyze the residual Pap test fixative by proteomic techniques for the discovery and verification of early-stage ovarian cancer protein biomarkers. Our central hypothesis is that proteins and small peptides shed by ovarian cancer cells can be detected in the residual Pap test fixative by mass spectrometry-based proteomics. 

Our scientific rationale for using the Pap test.
The Pap test fixative is ideal for the discovery phase of early-stage biomarker development since it is derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Several recent studies have shown that tumor-specific biomarkers are present at higher levels in biospecimens that are proximal to the tumor itself, as compared to blood samples. Recently, the fimbria of the fallopian tube has been suggested to be the true precursor to ovarian and primary peritoneal carcinomas, strengthening our hypothesis. As proof-of-principle, our preliminary data demonstrates that Pap test fixatives can be used to detect known ovarian cancer biomarkers in women with late-stage ovarian cancer; and furthermore, it contains fewer high abundance proteins, which have hampered biomarker discovery studies with blood samples.

The objective of this study is to discover proteins in Pap test fixatives that are unique, or present at higher levels, in Pap test samples from women with early stages of ovarian cancer compared to samples from women with benign gynecological conditions or healthy women. We will quantify the best candidate biomarkers in a second set of Pap test fixatives by using new state-of-the-art mass spectrometry technology. Future studies will focus on optimizing sample processing and assay development, as well as validation using larger cohorts.

The long-term goal of the OCEDP.
Our long-term goal is to develop an easy, noninvasive screening test that can be readily incorporated into a routine Pap test so that women can be tested simultaneously for cervical and ovarian cancer. The ultimate goal of this research is to develop a highly sensitive test for the detection of ovarian cancer in its early stages when treatments are most effective.

These OCEDP studies will impact women worldwide.
Results from studies being conducted at the OCEDP may be extended to the early detection of ovarian cancer in women in high-risk groups and eventually for screening the general population. We anticipate that these studies will shift the paradigm for current clinical tests and change the way that women worldwide are screened for ovarian cancer. Our "PapProtein" test has the potential to detect early stages of ovarian cancer when the disease is treatable; thereby increasing survival rates of women with ovarian cancer.

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Feasibility Studies

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics.

Boylan KL, Afiuni-Zadeh S, Geller MA, Hickey K, Griffin TJ, Pambuccian SE, Skubitz AP

Abstract: The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically "normal" results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate-polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database and characterized for localization, function and relative abundance.

View the full paper here.

Evaluation of the potential of Pap test fluid and cervical swabs to serve as clinical diagnostic biospecimens for the detection of ovarian cancer by mass spectrometry‑based proteomics

Boylan KL, Afiuni-Zadeh S, Geller MA, Argenta PA, Griffin TJ, Skubitz AP


Background: The purpose of this study was to determine whether the residual fixative from a liquid‑based Pap test or a swab of the cervix contained proteins that were also found in the primary tumor of a woman with high grade serous ovarian cancer. This study is the first step in determining the feasibility of using the liquid‑based Pap test or a cervical swab for the detection of ovarian cancer protein biomarkers.

Methods: Proteins were concentrated by acetone precipitation from the cell‑free supernatant of the liquid‑based Pap test fixative or eluted from the cervical swab. Protein was also extracted from the patient’s tumor tissue. The protein samples were digested into peptides with trypsin, then the peptides were run on 2D‑liquid chromatography mass spectrometry (2D‑LCMS). The data was searched against a human protein database for the identification of pep‑tides and proteins in each biospecimen. The proteins that were identified were classified for cellular localization and molecular function by bioinformatics integration.

Results: We identified almost 5000 proteins total in the three matched biospecimens. More than 2000 proteins were expressed in each of the three biospecimens, including several known ovarian cancer biomarkers such as CA125, HE4, and mesothelin. By Scaffold analysis of the protein Gene Ontology categories and functional analysis using PANTHER, the proteins were classified by cellular localization and molecular function, demonstrating that the Pap test fluid and cervical swab proteins are similar to each other, and also to the tumor extract.

Conclusions: Our results suggest that Pap test fixatives and cervical swabs are a rich source of tumor‑specific bio‑markers for ovarian cancer, which could be developed as a test for ovarian cancer detection.

Keywords: Ovarian cancer detection, Pap test, Mass spectrometry based proteomics, Biomarker

View the full paper here.

Microbiome Studies

Evaluating the potential of residual Pap test fluid as a resource for the metaproteomic analysis of the cervical-vaginal microbiome.

Afiuni-Zadeh SBoylan KLMJagtap PDGriffin TJRudney JDPeterson MLSkubitz APN.

ABSTRACT: The human cervical-vaginal area contains proteins derived from microorganisms that may prevent or predispose women to gynecological conditions. The liquid Pap test fixative is an unexplored resource for analysis of microbial communities and the microbe-host interaction. Previously, we showed that the residual cell-free fixative from discarded Pap tests of healthy women could be used for mass spectrometry (MS) based proteomic identification of cervical-vaginal proteins. In this study, we reprocessed these MS raw data files for metaproteomic analysis to characterize the microbial community composition and function of microbial proteins in the cervical-vaginal region. This was accomplished by developing a customized protein sequence database encompassing microbes likely present in the vagina. High-mass accuracy data were searched against the protein FASTA database using a two-step search method within the Galaxy for proteomics platform. Data was analyzed by MEGAN6 (MetaGenomeAnalyzer) for phylogenetic and functional characterization. We identified over 300 unique peptides from a variety of bacterial phyla and Candida. Peptides corresponding to proteins involved in carbohydrate metabolism, oxidation-reduction, and transport were identified. By identifying microbial peptides in Pap test supernatants it may be possible to acquire a functional signature of these microbes, as well as detect specific proteins associated with cervical health and disease.

Click here to view the full paper at PubMed.