Ovarian cancer is the uncontrolled proliferation of cells in the ovaries, the reproductive organ responsible for egg production in women. While it can impact women of all ages and ethnicities, mutations in a gene linked to breast cancer called the BRCA gene, as well as age, dramatically increase the risk of developing ovarian cancer, with post-menopausal women being the most vulnerable. 

“Ovarian cancer is a challenging disease to manage clinically,” explained Daniela Dinulescu, a lead researcher in ovarian cancer at Brigham and Women’s Hospital in Boston. “Most patients present with advanced stage and widespread metastases due to inefficient early detection and difficulty identifying precursor lesions and pinpointing their exact location. As a result, most patients typically relapse within two years following diagnosis.”

Due to this poor prognosis, ovarian cancer has a high mortality rate and is one of the leading causes of death among women with gynecological cancers.

To diagnose ovarian cancer, doctors currently combine imaging of the ovaries to visualize any abnormal tissue growth, along with a blood test looking for CA-125, a protein released by certain tumors, including those found in ovarian cancer.

However, these techniques are both unspecific, need well-established tumors and high levels of CA-125 for detection — all of these factors may not occur early on during the disease, limiting this diagnostic approach from effective, early detection. 

This prompted Dinulescu and a group of collaborative scientists from the Massachusetts General Hospital and the Brigham and Women’s Hospital in Boston, Massachusetts Institute of Technology in Cambridge, and the Institute for Basic Science in the Republic of Korea to seek for alternative means of detecting early signs of ovarian cancer that would allow life-saving, early diagnosis through a simple blood test.

The strategy

High-grade serous ovarian carcinoma (HGSOC) is the most common and deadliest form of the disease.  Dinulescu and others have demonstrated that although HGSOC tumors are found in the ovaries, they originate from skin cells within the Fallopian tubes, close to the ovaries. “The tubal precursor lesions, called STICs [serous tubal intraepithelial carcinoma], and seed onto the ovaries and [other organs in the abdomen],” explained Dinulescu. 

If tumors in the ovaries indicate advanced cancer, then diagnostic approaches should focus on their place of origin to be able to provide early detection. However, sampling cells in the Fallopian tubes is not so straightforward as it is highly invasive and can cause more patient trauma.

To avoid this, the team suggested an indirect way to assess Fallopian tube cells by analyzing extracellular vesicles — small, spherical, cell membrane-bound structures released by cells. They are found in a number of bodily fluids and transport various molecules, such as proteins, lipids, RNA and DNA, and other bioactive molecules, from one cell to another.

As extracellular vesicles carry components from their originating cells, they can serve as informers of health. The added advantage is that as they can be found in biological fluids, such as the blood, saliva, and urine, they allow for minimally invasive sampling (think a simple blood test or mouth swab), making them compelling tools for diagnosis and monitoring of cancer and other diseases.

“Extracellular vesicles produce functional information relevant not only for early detection but also relapse and drug resistance during treatment,” highlighted Dinulescu. “They provide excellent protection of their molecular cargo […] and can theoretically enable the detection of tumors less than 1 mm in diameter, or even smaller.”

The road to early testing  

In their recent study published in the journal Advanced Science, the team focused on detecting five proteins anchored in the membrane of extracellular vesicles released by cancerous cells in the Fallopian tubes into the blood. 

While a promising approach, the path was not straightforward as extracellular vesicles at the onset of the disease are typically present in low quantities in bodily fluids. To ensure accurate and sensitive detection, the scientists needed to improve on a classical method for measuring these proteins.

Normally, to study proteins on extracellular vesicles, scientists use special proteins called antibodies with the ability to stick to specific regions of cell structures. Antibodies in these tests have two primary purposes: first, they help attach the vesicles to a surface where scientists can do their tests. Second, labeled antibodies help find particular proteins in the vesicles, visualize them, and measuring their levels.

Attaching vesicles to the plate with antibodies often causes loss as antibodies are too specific, and some vesicles may not stick. To fix this, the scientists replaced the antibody-covered surface with a membrane that retains more vesicles. To further improve the test, they added a molecule called tyramide, which makes the signal of the labeled antibody stronger and allows for the detection of lower levels of proteins.

These modifications boosted sensitivity 1000 times; while the classical method needed 750,000 extracellular vesicles per sample to detect the proteins, the improved assay did it with just 600. This increase in sensitivity additionally reduced the sample volume needed to measure each protein.

The scientists next investigated which proteins were characteristic of vesicles released by cancerous Fallopian tube cells. Using their assay, they first identified 169 proteins found in these vesicles that could act as markers to identify them in diagnostic tests.

With the help of available protein databases and data gathered from the literature, the team was able to narrow the markers down to nine protein candidates related to cellular adhesion, immune response, and DNA repair, all processes of which are dysregulated in cancer and would act as a flag for the disease.

The team analyzed the nine markers in blood samples from mice and humans with ovarian cancer to see how well they correlated with the progress of the disease. In mice, all candidate proteins increased with cancer evolution; however, in clinical human samples, five followed the disease progression, narrowing the markers further to their top five candidates: EpCAM, CD24, HE24, VCAN, and TNC.

Testing the diagnostic power

In a clinical test, they assessed their test’s ability to diagnose and monitor cancer progression in a real setting. Through a blood test, they monitored the expression of their five proteins in 14 healthy volunteers as well as 37 patients diagnosed with ovarian cancer.

The team say they were able to detect ovarian cancer in 34 of the 37 ovarian cancer patients and accurately differentiate between healthy and affected individuals. However, their initial scoring system, which averaged the levels of the five markers, could not discriminate between the cancer stages as its values were similar for early and advanced cancer samples.

To solve this, the team looked at the expression levels of the five markers individually using a statistical method called linear discriminant analysis (LDA) that classifies samples with similar features into groups. For the purpose of this study, the features were the expression levels of the protein markers — each sample had five features, and the groups were designated the different stages of disease.

With this more advanced approach, they could differentiate between non-cancer, early stage, and advanced stage ovarian cancer samples.

Unveiling the clinical impact

The team hopes that in addition to improving clinical outcomes for patients with their early diagnostic test, they might also provide better cancer management and assist in decision making, especially for patients with BRCA genes.

“The standard clinical care for BRCA carriers consists of risk reduction surgery of ovaries between their thirties and forties,” explained Dinulescu. “However, this has a lot of side effects associated with surgical menopause, reduced quality of life, infertility, and long-term negative effects on cardiovascular disease and mental health.

“Early detection would allow high-risk patients to take a step-wise risk reduction surgery, first involving Fallopian tubal removal to preserve their ovaries during their reproductive years, and only remove ovaries closer to natural menopause.

“This new strategy, which is a major shift in the medical practice and cancer prevention, will have a big impact on reducing the side effects of surgical menopause,” concluded Dinulescu. 

Reference: Ala Jo, et al., Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection, Advanced Science (2023). DOI: 10.1002/advs.202301930

Feature image credit: fernandozhiminaicela on Pixabay