Invasive breast cancer is a disease that affects 1 in every 8 women  . Despite advances in screening tools and early intervention, it remains a common deadly disease, with many women experiencing progression or metastatic disease despite our best efforts at treatment. Although a few screening tools exist to determine who is at risk of recurrence, overall the molecular dynamics are still poorly understood  . It is also well described in the literature that angiogenesis plays an important role in hormonal resistance and further tumor recurrence in cases of previously treated breast carcinoma      . This understanding has suggested the use of angiogenic blockers as “preventive” therapies to be considered in early stages disease management. Unfortunately, these drugs’ side effects, and lack of longitudinal studies to support their application as prophylactic strategy, have been a barrier for their use in this fashion.
2. Methods and Materials
15 cases of Stage I-III breast cancer patients in whom tumors were estrogen receptor (ER) positive were randomly selected, and their biological cancer markers were assessed prior to, and after intervention. Most patients had undergone lumpectomy or mastectomy, and started hormonal blockade prior to the intervention. None of these patients received neoadjuvant chemotherapy or radiation.
Hormonal blockade was defined by intake of tamoxifen (dosed at least 10 mg per day, all had extensive metabolizer profile identified), or an aromatase inhibitor of physician’s choice. Several of these patients’ breast cancer assays performed by Oncotype Dx, and were given high recurrence scores (however, none of these patients chose to receive chemotherapy). The maximum Oncotype Dx score was reported at 50%. A liquid biopsy (circulating tumor DNA) was obtained when available. These were obtained prior to therapy, and performed again after therapy.
The antiangiogenic therapy was introduced by intravenous injections of a polyphenols including Quercetin, per multitargeted epigenetic therapy (MTET) protocol. The patients’ tumor markers, (such as CA 15-3 and CA 27.29), along with growth factors, and serum/plasma VEGF were monitored before, during, and after therapy. The hormonal blockade drug of choice in each patient was not changed. These patients did not receive any other mode of therapy, and were instructed not to change their diet or lifestyle.
100 percent of patients had reduced and/or normalized growth factors following treatment with the MTET protocol. These markers included fibroblast growth factor 2 (FGF-2), insulin-like growth factors 1 (IGF-1) and VEGF. In cases where liquid biopsy was available, there was complete resolution of circulating tumor DNA (ctDNA) post-therapy. There was direct correlation between the frequency of therapy and reduction in the biomarkers, specifically serum/plasma VEGF. The frequency of therapy was determined by elevations in these biomarkers prior to administration of the therapy. Patients with dramatically elevated biomarkers had treatment as often as ten treatments in a two-week period, while those slightly abnormal biomarkers were given treatment as infrequently as once a month. Since every individual’s biomarkers were measured and reported abnormal prior to therapy, every case represented their own control. There were no side effects reported from the therapy. None of the patients were found to have recurrence over the monitoring time, which so far has been between 6 months to 5 years. Here we include a case, with a summary of the results.
4. Case Study
A 68-year-old female with a history of left lobular invasive breast cancer, presented to our clinic treatment. She was first diagnosed in June 2016, and was status post biopsy of the left breast. The pathology showed ER and progesterone receptor (PR) positive, human epidermal growth factor receptor 2 (HER2) negative. A PET scan was done for staging, which was negative for distant metastasis. She had a remote history of thyroid carcinoma insitu, and was status-post thyroidectomy. She also had a history of laryngeal squamous cell carcinomain 2011, status-post radiation and cetuximab, which she responded to.
She was referred to us for second opinion, and was evaluated through BioFocus labs for circulating tumor cells (CTCs), Guardant 360 for ctDNA, and Caris Target Now for molecular profiling. Her molecular profiling revealed several mutations, including somatic mutations at BRCA (breast cancer susceptibility gene), CDH1 (cadherin-1), CSF1R (colony stimulating factor 1 receptor), IDH1 (Isocitrate dehydrogenase 1), and PI3k (phosphoinositide 3-Kinase) were detected by these labs. A BRCA germ line test was ordered and it came back negative. Tumor markers were drawn through LabCorp and were reported normal, although her serum HER2 was borderline normal at 14 (ng/mL). The CTC was also negative. Her ctDNA was positive for BRAF, AR, FGFR, and ERBB2.
She was started on Tamoxifen at 10 mg per day, as her Cytochrome P450 2D6showed an extensive metabolizer profile. She was started on daily IV epigenetic therapies (MTET protocol), which she received for 10 days over a two week period. Her serum Her2/Neu Quant level dropped from 14.6 ng/mL down to 11.7 ng/mL, post therapy. After 10 treatments, second ctDNA assay showed marked reduction of the mutant allele fraction (MAF), (see Figure 1), which AR and BRAF no longer detected, andFGF2 at 0.1%. The total MAF dropped from 1.4% down to 0.1%. The patient underwent mastectomy in August 2016. The surgical pathologyrevealed one of eleven lymph nodes positive, with positive lymphovascular invasion, and negative skin involvement. At that point, she was staged T3N1M0, with moderately differentiated lobular carcinoma. Her guardant was repeated post-mastectomy, which showed and increased in ctDNA. ERBB2, PI3k and ARID1, were detected, with a MAF of 0.8%. This increased prompted us to start the patient back on IV therapy with MTET protocol. Following treatment, her ctDNA was repeated in October 2016, and was found nondetectable.
Figure 1. Marked reduction of the mutant allele fraction (MAF).
This is an interesting case of successful response in a patient with no detectable distant metastases, but positive ctDNA. This demonstrates the concept of secondary prevention and reduced angiogenesis and heterogeneity of the tumor in response to MTET protocol, which consists of epigenetic therapies, as all ctDNA disappeared with the protocol. It is worth noting that neither tamoxifen nor mastectomy could positively impact the ctDNA, and in fact there was an increase in ctDNA post-mastectomy.
Our treatment protocol, which uses multitargeted epigenetic therapies, has generated interest in the use of combinational therapy to reduce recurrence, through a safe and effective method of treatment using natural compounds. We believe that such approach could change the current standard of care. Further research is needed to conclude whether this research would translate to improved progression free survival on a large scale, and to determine whether overall survival and cure rates are improved in the setting of hormonally sensitive breast cancer. Further trials are needed to optimize the therapy schedule and protocols.
 Ribatti, D., Nico, B., Ruggieri, S., Tamma, R., Simone, G. and Mangia, A. (2016) Angiogenesis and Antiangiogenesis in Triple-Negative Breast cancer. Translational Oncology, 9, 453-457.
 Bareschino, M.A., Schettino, C., Colantuoni, G., Rossi, E., Rossi, A., Maione, P. and Gridell, C. (2011) The Role of Antiangiogenetic Agents in the Treatment of Breast Cancer. Current Medicinal Chemistry, 18, 5022-5032.
 Coxon, A., Bush, T., Saffran, D., Kaufman, S., Belmontes, B., Rex, K. and Polverino, A. (2009) Broad Antitumor Activity in Breast Cancer Xenografts by Motesanib, a Highly Selective, Oral Inhibitor of Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors. Clinical Cancer Research, 15, 110-118.
 Heer, K., Kumar, H., Read, J.R., et al. (2001) Serum Vascular Endothelial Growth Factor in Breast Cancer: Its Relation with Cancer Type and Estrogen Receptor Status. Clinical Cancer Research, 7, 3491-3494.
 (2017) U.S. Breast Cancer Statistics.
 Qu, Z., Ginkel, S.V., Roy, A.M., Westbrook, L., Nasrin, M., Maxuitenko, Y. and Kern, F.G. (2008) Vascular Endothelial Growth Factor Reduces Tamoxifen Efficacy and Promotes Metastatic Colonization and Desmoplasia in Breast Tumors. Cancer Research, 68, 6232-6240.
 Liu, Y., Tamimi, R.M., Collins, L.C., Schnitt, S.J., Gilmore, H.L., Connolly, J.L. and Colditz, G. A. (2011). The Association between Vascular Endothelial Growth Factor Expression in Invasive Breast Cancer and Survival Varies with Intrinsic Subtypes and Use of Adjuvant Systemic Therapy: Results from the Nurses’ Health Study. Breast Cancer Research and Treatment, 129, 175-184.
 Cerezo, A.B., Winterbone, M.S., Moyle, C.W.A., Needs, P.W. and Kroon, P.A. (2015) Molecular Structure-Function Relationship of Dietary Polyphenols for Inhibiting VEGF-Induced VEGFR-2 Activity. Molecular Nutrition & Food Research, 59, 2119-2131.
 Reinert, T. and Barrios, C.H. (2015) Optimal Management of Hormone Receptor Positive Metastatic Breast Cancer in 2016. Therapeutic Advances in Medical Oncology, 7, 304-320.
 Traina, T.A., Dickler, M.N., Caravelli, J.F., et al. (2005) A Phase II Trial of Letrozole in Combination with Bevacizumab, an Anti-VEGF Antibody, in Patients with Hormone Receptor-Positive Metastatic Breast Cancer. Proceedings from the 2005 San Antontio Breast Cancer Symposium, San Antonio, December 2005.