Rationale: Women with localized or large breast tumors (cancer limited to the breast and/or lymph nodes) are often treated with a relatively new regimen called neoadjuvant therapy (NAT). NAT has a major advantage in that therapy before surgical resection (a) allows large tumors to shrink so that extent of surgery is limited, and (b) allows pathologic examination of the smaller tumor/tumor bed, enabling an objective assessment of how effective the neoadjuvant was on the tumor. Those who achieve a pathologic complete response (pCR) do better in the long run -- they have longer recurrence-free survival and longer overall survival. Currently, depending on the subtype, many women have an incomplete pathologic response and must go through additional therapy. So why do oncologists not continue treatment with the same or some other agent until the patient's tumor shrinks completely? This is because they have no way of telling if the tumor is shrinking or growing. Many groups are trying hard to develop "intermediate response markers" to address this unmet need. Testing blood for mutated DNA has been researched extensively, but gives sensitivity of detection of <40%, since particularly in the breast, there is a low frequency of common mutations and the tumors shed very little DNA into the circulatory system.

Methylated genes in breast cancer, on the other hand, are common, and we have reported that the pattern of methylation is retained from the primary tumor to distant metastasis, like a barcode for that tumor. Detecting tumor-specific methylated DNA in blood has required the development of techniques in our lab that can detect less than 50 copies in 300 µl of cancer plasma. In collaboration with the diagnostic company, Cepheid, we have converted this lab assay into an automated <5-hour assay called LBx-BCM, which is simple to perform and has a sensitivity of 88% and specificity of 92% for detecting Stage 4 metastatic breast cancer. In this Expansion Award, we would like to improve its performance and test it on patient samples so that it could serve as a reliable test for women and men undergoing NAT for Stage 3 breast cancer. In this way, we will provide a powerful tool to the oncologist to use as a surrogate for tumor load during NAT; to assess tumor shrinkage; to stop ineffective therapies; or to continue effective ones before surgery. More patients will thus achieve pCR and thereby longer time to recurrence and a longer lifespan.

Objectives: Our objective is to provide a sensitive and specific molecular blood test to the oncologist to assess response of the patient to the therapy at intervals during the neoadjuvant regimen. Our assay worked well in detecting and monitoring tumor load in Stage 4 metastatic breast cancer. But we know that the amount of DNA shed decreases with decreasing stage. Usually, the smaller the tumor, the less circulating DNA is shed by the tumor. So, our first objective is to increase the sensitivity of the cartridge test. We will test new ways to optimize DNA retrieval from plasma and test a newly developed 10-fluorophore detection cartridge, which will reduce cost and maximize use of the DNA (year 1). Our second objective is to test the performance of the optimized test. We will determine if methylation levels in blood measured using the LBx-BCM assay at two weekly intervals during NAT correlate with pathologic response in the surgically resected lesion. We will select one of these time points which best correlates with pathologic response for future work (years 2-3).

Ultimate Applicability: The target population for the application of the test developed in this proposal is women and men with Stage 3 cancer who will undergo NAT. Currently, there are no blood biomarkers to assess whether a patient is or is not responding to therapy, or whether an alternate therapy may be better. The endpoint is what the tumor looks like at the end of the treatment, upon surgical resection. It is critical to have a complete pathologic response, since long-term outcome is dependent on it. Subsequent treatment is given to patients who have incomplete or partial pathologic response, but there is no way of predicting the outcome. A reliable blood test can act as a surrogate for tumor load. Methylated DNA has a short half-life, and is stable, so repeated measures at short intervals can be made with confidence. The LBx-BCM cartridge assay can be performed by people with little expertise, and has few hands-on steps, so it can be implemented in any pathology laboratory. Optimizing the sensitivity of the assay, which is already very specific, will help not only patients with Stage 3, but eventually women with Stage 2 and Stage 1 breast cancer.

Overarching Challenge: Quantitative measurement of circulating methylated DNA that would reflect tumor burden in real time is essential, and the use of LBx-BCM assay for this purpose is promising. It is time to address the overarching challenge of "Developing reliable, quantitative, specific, and sensitive liquid biopsy biomarkers to continue effective therapies or to discontinue ineffective ones in a timely manner."