In spite of years of research and improvements in breast cancer therapy, each year 40,000 women in the US and 600,000 women worldwide die of metastatic breast cancer.

In most women, a complete cure of the local tumor is achieved through surgery and radiation. However the tumor recurs early or late as metastasis at distant organs. Currently there are no reliable blood tests that can be used for nearly all women with metastatic breast cancer. The CTC (circulating tumor cells) test, for example, is a good test, but tumor cells are detectable in blood in only in about 40% of the women. Other tests like mutation detection in blood are expensive and rely on information that has to be collected by sequencing the primary breast tumor that was resected many years ago, and old blocks are often difficult to access from far-away hospitals.

What can blood tests do for women with metastatic breast cancer? Molecular tests that detect cancer DNA floating in the blood could be very useful. A positive test could alert the physician that the disease has returned. They can also act as red flags (cancer indicators) to determine if the patient is, or is not, responding to therapy (prediction of response). Rapidly arriving at the best effective drug can reduce suffering due to side effects, save costs of ineffective therapy both to the person and to their finances, and hasten the path to recovery while the tumor burden is small. Further, the test can provide a “crystal ball” prediction as to whether the disease will regress or come back in the future (prognosis).

The overarching challenge addressed by this proposal is to reduce morbidity and mortality associated with metastatic breast cancer. Reliable and rapid tests for the prediction of response to an initiated therapy and prognosis of the outcome of metastatic breast cancer are sorely needed. Blood tests that provide an accurate reflection of disease burden will minimize side effects and delay from ineffective therapy, reduce costs from additional imaging studies, and provide longer tumor-free, healthier lives to women with breast cancer.

For the past 20 years, we have worked on a DNA change called “methylation” in certain genes that happens in tumors but not in normal breast tissue, or in white blood cells. We developed tests to perform tissue measurements of multiple methylated genes from the same small numbers of tumor cells -- this was relatively easy. But detecting methylated DNA in the blood was very difficult since a very small amount of tumor DNA is shed into the blood, and finding this “needle in the haystack” of normal DNA shed by tissues required us to develop special highly sensitive techniques. We developed a technique (cMethDNA) and tested it thoroughly in the lab using serum from women with metastatic breast cancer. The test could detect cancer DNA floating in the blood in more than 90% women (sensitivity: how many cancer cases does it detect?), and with higher than 90% specificity (how often can it differentiate between blood from cancer versus normal?). Now we are ready to convert the lab test into an automated test that can be run on a machine by people with minimal training. This proposal is focused on studies that will clinically translate this blood test to an automated 4-hour test that can be performed with ease worldwide to predict response rapidly. Our aims are designed to: (1) optimize the functioning of the test, to make sure that the results of the cartridge assay are as good or better than cMethDNA, our lab assay; (2) test the cartridge using serum from patients with breast cancer that are divided into two groups called training and test sets; and (3) validate the utility of the test for predicting response to therapy and provide prognosis information by testing sera from 141 patients collected as soon as they started treatment and at intervals during treatment. We will compare the efficiency of this test to our cMethDNA assay and to those obtained by the circulating tumor cell (CTC) assay on the same patients. Our projected time to testing the cartridge as described in this proposal is no more than 3 years. All the serum samples are already collected, so no lag time is expected once the cartridge assay is validated. If the test passes all this scrutiny, in the future, we will put the test through even more rigorous testing by collecting blood from a large trial (about 500 women) on metastatic breast cancer (blood collection will be initiated in year 2) in readiness for requesting Food and Drug Administration (FDA) approval for its use.

The impact of this study on improving the health and outcome of women with breast cancer metastasis will arise from two developments. First, the emergence of many novel therapies, cell cycle inhibitors, heat shock protein inhibitors and DNA damage-, and immune checkpoint-inhibitors that are already happening. Second is the ability to quickly go through the available armamentarium of therapies to find which specific one the metastatic tumors respond to, and most effectively. Our test will have the ability to achieve this goal. High DNA methylation in the blood measured at 2-3 weeks after starting a therapy is bad news, suggesting that the tumor is continuing to grow, while low or no methylated DNA in the blood is excellent news, indicative of the reduction in metastatic tumor burden. Further, if there is no tumor-DNA in the blood within 3-4 weeks of starting treatment, the woman will enjoy a longer recurrence-free life and will live longer as well. Thus, our test will be able help to reduce morbidity (side effects) and mortality in women with metastatic breast cancer.