Utilizing Tumor and Plasma Liquid Biopsy in Treatment Decision Making for an Estrogen Receptor-Positive Advanced Breast Cancer Patient

Breast cancer affects 12% of females in the United States and is the leading cause of cancer death in the female population. Personalized therapy is being used in clinical practice to treat breast cancer based on tumor molecular profiling, which can be obtained from tissue biopsy or plasma liquid biopsy as circulating tumor deoxyribonucleic acid (ctDNA). The available ctDNA tests provide a non-invasive way to monitor the cancer genome in a real-time manner. In this case report, a 38-year-old female with recurrent estrogen receptor (ER) positive breast cancer is treated with letrozole, everolimus, and palbociclib. The drugs target the hormonal signaling pathway, phosphoinositide 3-kinase (PI3K)-RAC-alpha serine/threonine-protein kinase (AKT) pathway, and cyclin D1 (CCND1)-CDK4/6 pathway, based on the patient’s estrogen-receptor-positive (ER+) disease and phosphatidylinositol -4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation, as well as PIK3CA and CCND1 amplification. After 11 months of treatment, retinoblastoma protein transcriptional corepressor 1 (RB1) mutation was caught in ctDNA, which suggests an acquired resistance to palbociclib. Pazopanib was then used instead of palbociclib, targeting the fibroblast growth factor 3/4/19 (FGF3/4/19) amplification that was initially observed in her molecular profiling. Trametinib was also suggested recently due to the increasing allele frequency of B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutation in ctDNA, following the treatment of letrozole + everolimus + pazopanib. The patient has no evidence of disease after five months of treatment initiation and has remained disease-free for over 16 months. In conclusion, the analysis of ctDNA is an effective way to monitor the real-time changes in a patient's tumor genome, which is a great supplement to the molecular profile from the tissue biopsy. The combination of these two tests provides an efficient strategy to make more informed treatment decisions, which greatly adapt along disease development.


Introduction
Breast cancer is a leading cause of cancer death in females worldwide [1]. According to the United States breast cancer statistics, about 12% of women are affected by the disease in America. Among breast cancer patients, over 70% of them have estrogen receptor (ER) positive 1 1 1 1 1 1 1 disease. After decades of investigations, the research communities have a far greater understanding of the disease at the molecular level, especially with the identification of genetic alterations related to the development of breast cancer. With the continuous development of sequencing techniques in recent years, personalized treatment plans are in practice based on molecular profiling for the specific patient.
Among breast cancer patients, molecular profiling is normally performed using a biopsy from either primary or metastatic tumor tissue. However, the genetic changes of cancer are not stable but dynamic. It has been known that cancer is a genetic disease. This results in heterogeneous tumors that change continuously throughout the course of the disease. Treatment plans need to be adjusted according to the changes. But in practice, obtaining biopsy from patients frequently to update their molecular profiles is not practical.
With the development of the liquid biopsy and sequencing using circulating tumor deoxyribonucleic acid (ctDNA) from plasma samples, a new approach is providing the opportunity for closer monitoring of disease development. Plasma ctDNA is obtained in a noninvasive way as a blood draw, which allows clinicians to have a real-time evaluation of the tumor burden, discover emerging genetic alterations and change treatment regimen in a timely manner.
In this case of ER-positive metastatic ductal carcinoma of the breast, we found dynamic changes in ctDNA, including changes in allele frequency of existing mutations and presenting of new genetic alterations. The treatment plan was originally made based on the molecular profile of the tumor biopsy and changed based on ctDNA findings. Based on this strategy, the patient has remained disease free for over 16 months after recurrence. Informed consent statement was obtained for this study.

Case Presentation
A 38-year-old Caucasian female was seen in our clinic in August 2015 with a recurrent disease of the breast in the left supraclavicular lymph node.
The patient was first diagnosed in 2011. The tumor was grade 3 (3/2/3) by Nottingham grade; estrogen receptor (ER) positive, progesterone receptor (PR) positive and human epidermal growth factor receptor 2 (HER2) negative by immunohistochemistry (IHC). Right breast mastectomy and axillary lymph node dissection were performed with left breast prophylactic mastectomy. Final pathology suggested a stage IIB (T2N1aM0) invasive ductal carcinoma according to the Union for International Cancer Control (UICC) Tumor Node Metastasis (TNM) classification for breast cancer. The tumor measured 4.4 cm. Two out of nine lymph nodes were positive for cancer invasion. The patient was treated with dose-dense doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) after surgery and completed post-mastectomy radiation therapy to the right breast in May 2012.
The patient was then maintained on estrogen blocker (tamoxifen) and ovarian suppressor In addition, the same biopsy was tested through FoundationOne® for genomic profiling. FoundationOne® is a comprehensive genomic profiling assay, sequencing the coding region of 315 cancer-related genes and introns from 28 genes to a median depth of coverage of 500X. Alterations detected by FoundationOne® are presented in Table 1. DNA repair associated breast cancer 1/2 (BRCA1/2 ) test from Comprehensive BRACAnalysis® (MYRIAD Genetic Laboratories, Salt Lake City, Utah ) came back with negative results.

Gene Effect
(amino acid change) Gene Effect (copy number change, rearrangement and insertion)  Based on the patient's disease status, tumor marker (ER/PR/HER2) and genetic testing results, we started the following regimen with letrozole (2.5 mg daily), palbociclib (75mg day one through day 21 of a 28-day cycle) and everolimus (5 mg    An illustration of disease progression and treatment for this patient is presented in Figure 1.

Discussion
In the case described above, after disease recurrence, the next line treatment recommendation was made based on the molecular profile from the recurrent tumor biopsy at the patient's lymph nodes as well as her tumor marker results.
Since the disease was ER positive, letrozole was selected first in the regimen as an aromatase inhibitor to lower estrogen level. One of the most commonly modified pathways in ER-positive breast cancer is phosphoinositide 3-kinase (PI3K)-RAC-alpha serine/threonine-protein kinase (AKT) pathway. Activation of this pathway is associated with resistance to hormonal therapy, while promising results have been suggested by clinical trials using the combination of endocrine therapy and PI3K-AKT pathway inhibitors [2]. PIK3CA encodes the catalytic subunit of PI3K. Both PIK3CA amplification and mutation, as detected in this patient are associated with an enhancement of downstream signaling and over-activation of Akt. Akt blocks mammalian target of rapamycin complex 1 (mTORC1) inhibitor tuberous sclerosis proteins 1 and 2 (TSC1/2). Without TSC1/2, mTORC1 is over activated and phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K), which increase translation and promotes cell proliferation. Everolimus was added to the regimen as an mTORC1 inhibitor, targeting the PIK3CA aberrations found in the patient [3].
Another pathogenic alteration suggested by the patient's molecular profile is CCND1 amplification. The vast majority of CCND1 amplified breast cancer are reported to be ER-positive and associated with poor prognosis [4]. In estrogen-driven breast cancer, oncogenic signaling through estrogen stimulated the CCND1-CDK4/6-dependent retinoblastoma protein (Rb). CCND1-CDK4/6 complex phosphorylates Rb and releases transcription factor E2F (E2F) from the Rb-E2F complex. Once E2F is released, it can switch cells from G1 to S phase and start cell proliferation. This proliferative stimulus was augmented by amplification of CCND1. Palbociclib was included in the regimen as a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor to block the phosphorylation of Rb and inducing G1 arrest.
However, a good clinical response of palbociclib relies on a wild type, functioning Rb protein.
Loss of Rb function was reported in different cases to relate with palbociclib resistance [5][6].
When RB1 mutation was caught in ctDNA from this patient, palbociclib was taken off from the regimen.
Another potential target in the molecular profile from tumor biopsy was fibroblast growth factor 3, 4 and 19 (FGF3/4/19), which indicated an active ancillary angiogenic pathway. Pazopanib was introduced to the treatment plan when palbociclib was stopped, to prevent treatment resistance of palbociclib and further block the activities of the FGFs. Upon the binding of FGFs to the FGF receptors (FGFR), FGFRs forms dimers. The dimerization process activates FGFR kinase activity. Activated FGFR kinase further phosphorylates and activates different pathways, which promotes cell proliferation, survival, and motility [7]. Pazopanib, as a pan-tyrosine kinase inhibitor including FGFR, is proven to be clinical beneficial from different trials for breast cancer [8][9].
Although the patient remained disease free on the treatment, the consistent and rising BRAF (p.Phe595Leu) mutation in ctDNA caused concerns. BRAF mutations in the kinase domain can result in an elevated kinase activity, which directly phosphorylates and activates serine/threonine protein kinase MEK1/2 (MEK1/2) on the Ras-Raf pathway. The over-activation of this Ras-Raf pathway can further induce a series of Elk-dependent transcription and increase cell growth and proliferation [10]. In order to prevent tumor recurrent from the BRAF mutation, trametinib will be started in the next cycle as an MEK1/2 inhibitor to block the Ras-Raf pathway.
The rationale of the regimens is illustrated in Figure 2 and abbreviations are listed under Table  3.

Conclusions
Based on the initial molecular profile from a tumor biopsy, we are able to restrict the disease progression and no evidence of disease is suggested on PET/CT after five months of targeted therapy. Also with the information provided by a sequence of ctDNA testing, the changes of tumor genome are closely monitored and the treatment regimens are adjusted accordingly in a timely manner. Benefited from both tumor tissue and ctDNA sequencing, a series of personalized treatment plans are made for the patient, aiming at targeting the drivers of the disease precisely and prevent acquired treatment resistance at the best possibility. As a result, the patient is successfully maintained in disease-free status for over 16 months.
We are planning to follow the treatment protocol, re-check ctDNA and imaging every three months and change the regimen when necessary. With this plan, we are confident to maintain the patient in disease-free status without further cancer progression.