Circulating Cell-Free DNA and Breast Cancer Biopsies: The Liquid Approach

The advent of the so-called liquid biopsy in recent years has garnered significant attention and research within the field of oncology. And rightly so. The benefits of this approach to research and clinical practice are numerous, and the literature has seen a considerable increase in published findings where the measurement of circulating tumor cell-free DNA (cfDNA) within studies has played significant roles in understanding aspects of basic cancer biology, disease progression, and response to treatment modalities.

Less invasive by way of blood draws, analyzing a patient's circulating cf DNA affords the ability to readily biopsy metastatic disease that has infiltrated difficult-to-reach areas, such as the brain, bones, liver, and other delicate structures. The technique allows for a much easier way to longitudinally biopsy these patients in a way that is simply not practical, desirable, or safe using traditional methods. "The approach allows us to obtain a snapshot in time of the patient's complete tumor picture. For example, in the case of metastatic disease, with one blood draw we can see aspects of different tumors in different locations within the body through the unique signatures and markers of their cf DNA," says Adrian Lee, PhD, Pittsburgh Foundation Endowed Chair in Precision Medicine, professor of Pharmacology and Chemical Biology, and co-leader of the Hillman Breast and Ovarian Cancer Program at UPMC Hillman Cancer Center. 

Dr. Lee's translational research is focused on several primary areas related to understanding and targeting the insulin-like growth factor pathways in breast cancer, including the role of ESR1 mutations seen primarily in relapsed and metastatic disease. In recent years, Dr. Lee and colleagues have become interested in using the detection of circulating cfDNA in their studies of ESR1 mutations in primary, relapsed, and metastatic breast cancer.

The first of their studies on this topic, the findings of which were published in 2016 in the journal Clinical Cancer Research, showed several unexpected results and subsequently led to further studies and a phase II clinical trial currently in progress.

Analyzing ESR1 Mutations in Breast Cancer

Estrogen receptor alpha and the ESR1 gene are implicated in most instances of breast cancer, and ESR1 mutations have been shown to be significant with respect to metastatic and relapsed cases of breast cancer, and indicative of, or possibly driving, treatment-resistant disease conditions. Three years ago, as research was coming out showing mutations of ESR1 to be important indicators of advanced disease and disease refractory to endocrine therapy, Dr. Lee's group began to take interest in the findings and designed a study to see what they could find in a small cohort of active patients from the breast cancer clinic at UPMC Hillman Cancer Center using newly acquired ddPCR (droplet digital polymerase chain reaction) technology that allows for incredibly sensitive and specific quantitative data regarding cfDNA findings within a sample. 

"We enrolled 40 randomly selected women with advanced breast disease into the study, taking serial blood draws over time to see what we might find in terms of ESR1 mutations," says Dr. Lee. The study also examined samples from primary tumors and brain metastases derived from the large, onsite tissue bank at the University of Pittsburgh. The results were a bit surprising to Dr. Lee and his collaborators, and also indicative of just how fast the science is progressing in the field. 

Primary tumors showed the smallest frequency of ESR1, mutation at 7 percent. Bone and brain metastases samples showed mutation rates of 9.1 percent and 12.5 percent, respectively. In the cfDNA samples, mutations of ESR1 were found in 24.1 percent of the population. "With our research and that of others, it's clear that these ESR1 mutations are important. But, we still don't know exactly what having any of the mutations means relative to their specific influences over disease state, and what a corresponding clinical indication would be in terms of therapy. This is where our current clinical trial picks up on these findings--beginning to look at the clinical implications," says Dr. Lee.

This trial² is a phase II examination of cases of metastatic breast cancer being treated with either fulvestrant or tamoxifen after having had a previous treatment with an aromatase inhibitor. Patients will be randomized into one of two treatment arms and administered either the fulvestrant or tamoxifen, and both will be tested via cfDNA examinations for ESR1 mutations prior to and after treatment.

"Our goal is to do basic science that gets into the clinic. This research is a great example of starting from scratch and rapidly progressing to the clinic in just a few short years. It's rare for us that our research progresses at such a pace, but it is indicative of just how fast the science is moving and what kind of promise it holds for bettering our understanding of this disease in its many forms."

Future Applications for cfDNA

The use of the liquid biopsy for measuring and detecting circulating cfDNA from tumors may, in the future, also extend to surveillance of individuals who are at high risk for developing breast cancer, or monitoring for signs of recurrent disease in individuals who have had a first line therapy or surgical resection of a primary tumor. There is some data in the literature now showing that women with detectable levels of cfDNA six months or a year after surgery have a higher risk for relapse than if they did not. "As a surveillance mechanism, I think a lot of people feel this approach holds great promise. It is a very personalized approach, because we would know in advance the mutations in the tumor the patient had and can look for it after the fact. We do this at the moment with cancer antigens, but they are very nonspecific and generally don't work well," says Dr. Lee.

The ability to detect changes at the molecular or DNA level within a patient's blood, before even the most sensitive imaging tests could detect the presence of a primary tumor or likely formation of one, or before the manifestation of symptoms occur signaling disease, could allow for greater precision of treatment at a time when the malignancy is in its earliest state of formation. "Because of the sensitivity and specificity of this kind of test, we may be able to use the approach for early detection of cancer in individuals with known genetic predispositions or other high-risk scenarios, such as women with dense breast tissue. This kind of routine clinical application of the testing platform is in the future, but the potential is there if we know what biomarkers or DNA mutations to look for in these individuals. It all depends on what the individual's cancer looks like," says Dr. Lee.

Much more research is needed before such an ability is possible, but it's the possibility of these sorts of advances that continues to drive Dr. Lee and his research colleagues to pursue and unlock the complex mysteries of cancer development and progression.

"None of these advances are or would be possible without the work of so many researchers and clinicians, and of course our patients who literally devote parts of themselves to help further our research. Our work is for them but would not be possible without them."

References and Further Reading
1. Wang P, Bahreini A, Gyanchandani R, Lee AV, Oesterreich S, et al. Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell Free DNA of Breast Cancer Patients. Clin Cancer Res. 2016; 22(5): 1130-1137.
2. Phase II Treatment of Metastatic Breast Cancer With Fulvestrant or Tamoxifen. ClinicalTrials.gov Identifier: NCT02913430. 
3. Gyanchandani G, Kota KJ, Jonnalagadda AR, Minteer T, Knapick BA, Oesterreich S, Brufsky AM, Lee AV, Puhalla SL. Detection of ESR1 Mutations in Circulating Cell-Free DNA From Patients With Metastatic Breast Cancer Treated With Palbociclib and Letrozole. Oncotarget. 2017; 8(40): 66901-66911.