The Active Ratio Test: A multiple-biomarker blood test for ovarian cancer early detection 

For over a decade the OCRF supported Dr Stephens’ early detection research, which has since led to a clinical trial of their new ovarian cancer diagnostic test. As there is no current early detection test, nor a non-invasive and reliable diagnostic approach for ovarian cancer, this is important research.  

The test measures levels of multiple biomarkers, or indicators of disease, which collectively signal whether ovarian cancer is present or absent.  

Funded by the OCRF, Dr Stephens’ team identified one of the biomarkers, CXCL10, as a promising biomarker of ovarian cancer, and excitingly, very early-stage ovarian cancer.  

By examining the profile of proteins within ovarian cancer samples and comparing it to the profile of proteins in healthy samples, the team identified approximately 100 different proteins present in the cancerous samples that were not in the healthy controls. One of the differences they observed was in a protein called CXCL10. Over several years they looked at how CXCL10 is changed in ovarian cancer, and how this can affect its function.  

This project will enable Dr Stephens and his team to collect data towards a diagnostic test for ovarian cancer and develop a multi-marker test incorporating the measurement of CXCL10. 

How does the test work? 

• It measures the levels of multiple biomarkers including, importantly, two forms of the CXCL10 protein: One of the earliest OCRF grants Dr Stephens received was for mass spectrometry equipment, which can measure and identify tiny molecules or proteins in samples. This allowed him to demonstrate that the CXCL10 in ovarian cancer is different to normal CXCL10 and could potentially be used to indicate early ovarian cancer.  
• This work formed the basis of the new test, which: Measures the ‘active’ and ‘total’ forms of CXCL10, CA-125, HE4 and IL-6. An algorithm combines those measurements into a single score to indicate the risk that an ovarian abnormality might be cancerous. 
• It measures a specific modification of CXCL10 proteins that only occurs in the early development of cancers, not in non-cancerous conditions: At the earliest stages of ovarian cancer tumour development there is an inflammatory response at the location where the tumour is going to form. Normally, the job of CXCL10 is to recruit immune T-cells to help stop the cancer from forming. However, when they are first developing, ovarian cancers produce enzymes called Dipepitidyl peptidases. These enzymes modify the structure and function of CXCL10 and ‘switch off’ the ability for CXCL10 proteins to recruit immune cells and blocks the ability of other T-cells to respond. This is one of the ways that ovarian cancers can ‘hide’ from the immune system while in the early stages of development.  

Because the test is based on a change to a protein that only occurs in cancer, and only at the very earliest stages, the team believe that it holds promise for both accurate diagnostics and early detection. 

Testing how accurately and how early the test can detect ovarian cancer  

Dr Stephens and his team collaborated with top researchers and clinicians across Australia and New Zealand (Aotearoa), led by Professor Tom Jobling and Professor Martin Oehler, to collect samples, particularly from individuals at a high-risk of ovarian cancer as they may have the earliest, pre-cancerous, signs of the disease. These included samples from individuals at high-risk of ovarian cancer who were having prophylactic, or preventative, surgery and consented to samples being collected for the research. 

Dr Stephens’ diagnostic and early detection approach holds promise as demonstrated by the below outcomes of OCRF funding: 

Diagnostics: in approximately 300 samples the test was able to accurately identify ovarian cancer more accurately than ultrasound, which is standard practice for diagnosis. 

Early Detection: in approximately 275 patients the test could identify ovarian cancer more accurately (with 91% specificity) and earlier (with 90% sensitivity) than currently available methods. Additionally, this study showed the test could identify ovarian cancer in some pre-cancerous samples, indicating a potential ability to detect ovarian cancer development before it even becomes cancer. 

The test has now been licensed to Australian company Cleo Diagnostics Ltd, an ASX listed company, who are taking it through the US Food and Drug Administration (FDA)  regulatory approval process, aiming for market entry in 2026. 

“OCRF funding has been absolutely critical to the success of this work. The OCRF provided sustained funding from the very early stages.” 

Trials and next phases 

With OCRF funding now complete, Dr Stephens is working with Cleo Diagnostics to progress this diagnostic and early detection approach towards three tests that will use the same foundational technology and science but serve three different purposes. Before being used in the clinic, each test will be trialed as below and, if successful, made available in the following order:  

• The Cleo Pre-Surgical Triage Test: This involves a clinical trial currently underway in the USA. Sixteen centers across the US are recruiting a minimum of 500 women identified with an ovarian growth requiring surgery. The trial is focused on accuracy of testing, with the results used to determine the risk that an ovarian cancer is present or not. Such a test would assist in directing patients to the most appropriate care, enabling rapid treatment and optimising medical resources. This trial will enable the team’s FDA application, with data collected in compliance with the FDA’s regulatory framework. This important trial is the critical step in gaining approvals for the test to enter clinical use. To be eligible, patients need to have had an ultrasound, a mass identified, and be booked for surgery. Before surgery, Dr Stephens triage blood test will be completed. The current prototype of the triage test holds 95% sensitivity and 95% specificity for detecting ovarian cancer accurately and distinguishing it from benign conditions. 
• Recurrence Test: This test will monitor women who have already been treated for ovarian cancer, to ensure that any recurrence is identified early. This aims to give clinicians as much time as possible to develop the most effective treatment approach for patients if the cancer returns. “This trial will have a smaller number of patients than the current trial, but will run for longer due to the need to monitor patients over time,” said Dr Stephens. 
• Early Detection Screening Test: Screening to identify early-stage cancers is the best way to significantly improve survival outcomes. This test will likely be rolled out in two phases, with the first focused on those women deemed at “high-risk” of developing ovarian cancers. This includes women with strong familial histories or hereditary genetic predisposing factors (e.g. carriers of BRCA1 and BRCA2 mutations). The second phase will seek to develop a general population screening test.  Both phases will require very large numbers of women (likely in the tens of thousands), and the team are currently investigating and preparing for the statistical requirements.  

The outcomes of this OCRF funding have contributed to the design and development of ovarian cancer testing towards clinical translation, with significant potential to improve diagnosis and pre-surgical evaluation for patients. This could improve patient care, overall treatment strategies/outcomes, as well as the allocation and use of medical resources. These tests hope to ensure individuals who do have ovarian cancer receive a faster diagnosis and appropriate care rapidly.  

Currently the test is focused on epithelial ovarian cancers, the most common type of ovarian cancer. This includes the serous (high and low-grade), mucinous, endometroid, clear cell, and granulosa cell types of ovarian cancer.  They hope to expand the test to include rare ovarian tumours in the future.