Triple negative breast cancers compromise about 15% of breast cancers overall, about 70% of breast cancers in individuals harboring a germline BRCA1 mutation, and 20% in BRCA2 mutation carriers, vice versa, the presence of germ line and/or somatic mutations in BRCA has been widely recognized in triple negative breast cancer. As of January 2016, there were more than 6 PARP inhibitors under development for BRCA positive tumors (See Table 1).
That said, a common failure of phase III trials has been improved progression free or more importantly the overall survival in the group of patients with triple negative breast cancer treated with single PARP inhibitors. A good example of this is phase III trial using Inaparib in patients with triple negative breast cancer who also received cytotoxic chemotherapy and showed no improvement in survival (phase II trial however showed promising results). A common rationale on the failure of the drug is the epigenetic aberrancies involving the BRCA gene and acquired resistance explained by secondary mutations.
In Oct., 2016, the preclinical work of Dr. Baylin at John Hopkins showed that PARP inhibitors work according to how intensely and durably the PARP
Table 1. List of all PARP inhibitors.
enzyme is trapped at certain DNA damage sites. Therefore, by ramping up the duration and intensity of this trapping, you could potentially increase the efficacy of the drug. Such understanding provides the rationale on combination of epigenetic therapies with PARP inhibitors   . That said, to our knowledge, the combination of epigenetic therapies consisting of the protocol we used with a known PARP inhibitor has never been clinically explored in an advanced case of triple negative breast cancer and clinical relevance been discussed.
Epigenetic therapies, consisting of polyphenol, Quercetin as mainstay of therapy, and phenyl butyrate, in a protocol called “multitargeted epigenetic therapies” administered intravenously on daily basis.
3. Case Report
37 yr female w/metastatic breast ca to bone, lymph nodes, and brain, she was Caucasian, had positive family history for maternal breast cancer, and was non smoker.
Patient was initially diagnosed in July 2013 w/stage III right breast ca and 4 axilla lymph nodes. BRCA 1w/Met ER+. Pt received 4 cycles of ATC, then underwent double mastectomy, followed by 36 rounds of radiation therapy, and then prophylactic oophorectomy.
In June 2015, CA27/29 marker noted to be elevated and scans indicated mets to left lung and left clavicle lymph nodes. Pt participated in UCLA trial w/PARP inhibitor from July to October 2015. Then switched to Xeloda from Nov. 2015 to Jan. 2016. From Feb. to March 2016 patient was on Aromatase inhibitor and Ibrance. From April to May 2016 patient received Carbo/Gemzar but during this time period patient developed pain in sacrum. In June 2016, she received radiation to sacrum with relief of pain and also received radiation to two brain lesions per cyberknife. In July to Aug. 2016 participated in Stanford trial BMO1103 and received more radiation to another brain lesion.
She also received 10 rounds of radiation to lesions noted on neck. In Oct. 2016, patient started immunotherapy, PARP, and Ameridex. Her restaging PET scan showed very large mass in the left lower lung, as well as severe lymphadenopathies in her neck, thorax and pelvis, retroperitoneum with mass effect on IVC, causing hydronephrosis and partial colon obstruction, due to peritoneal carcinomatosis, along with sacrum large lesion with significant uptake, compared to her PET scan in 5/15 (see Image 1).
She was referred by her oncologist to us for evaluation and treatments. On her initial evaluation, she was in significant pain. Labs indicated 93 percent MAF of c DNA with several alterations, including BRCA. CTC was positive for three out of four markers, very extensively high.
Immediately after starting the multi targeted epigenetic therapies (MTET), she was feeling better and breathing better and had more sense of well being after
Image 1. Whole body PET scan
the first two treatments. Pain was better. Less nauseous. More appetite. She did not experience any toxicity from the treatment.
Lab showed significant reduction in her tumor markers (CA 27.29 and CA 15.3) and LDH, measured on 11/22/16, compared to 11/7/16.
CA 15.3 at 179 from 249. LDH down from 1603 to 1348 and further down to 1298 (measured on 12/6/16) and 890 two weeks after. After two weeks of therapy all her CTC markers dropped including the CK19, telomerase, ERBB2 and c Myc.
Figure 1. Circulating tumor cells.
This happened without using any chemotherapy (please see Figure 1).
Her circulatory DNA through Guardant dropped from 93 percent on BRCA to non detectable (See Figure 2). This substantial response confirms the synergistic effect of the applied combinational therapy, and noticed in only 14 days after the initiation of the therapy.
Further the PARP inhibitor was stopped and the polyphenol therapy was continued. The c DNA was repeated on 12/20/16 and it showed increased BRCA back up again at 86 percent (please see Figure 3).
Patient accomplished unexpected response to the combinational therapy of PARP inhibitors and epigenetic protocol, with improved progression free survival and quality of life. She was under our care for three months and did not encounter any toxicity form the therapy.
Application of dual inhibition of epigenetic modifier consisting of natural compounds implemented in multitargeted epigenetic therapy (MTET) and traditional PARP inhibition is feasible and clinically relevant. Such therapy in our study showed significantly effective in treating BRCA positive colonies in tumor in triple negative breast cancer. The correlation of liquid biopsy findings and clinical response to such combinational therapy is also important as it can be used as a companion diagnostic for the therapy. We conclude that such therapy could replace the current standard of care in advanced triple negative breast cancer harboring BRCA deficient somatic or germ line mutations.
Figure 2. Circulating DNA (11/22/2016).
Figure 3. Circulating DNA (12/20/2016).
 Veliparib Falls Short in Phase III NSCLC, TNBC Trials (2017)
 Dziadkowiec, K.N., Gasiorowska, E., Nowak-Markwitz, E. and Jankowska, A. (2016) PARP Inhibitors: Review of Mechanisms of Action and BRCA1/2 Mutation Targeting. Przeglaad Menopauzalny/Menopause Review, 15, 215-219.
 Oshaughnessy, J., Schwartzberg, L., Danso, M.A., Miller, K.D., Rugo, H.S., et al. (2014) Phase III Study of Iniparib Plus Gemcitabine and Carboplatin Versus Gemcitabine and Carboplatin in Patients with Metastatic Triple-Negative Breast Cancer. Journal of Clinical Oncology, 32, 3840-3847.
 Muvarak, N.E., Chowdhury, K., Xia, L.M., Lapidus, R.G., Baylin, S.B. and Rassool, F.V. (2016) Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents: A Potential Therapy for Cancer. Cancer Cell, 30, 637-650.
 Han, H.S., Diéras, V., Robson, M., et al. (2016) Efficacy and Tolerability of Veliparib (V; ABT-888) in Combination with Carboplatin (C) and Paclitaxel (P) vs. Placebo (Plc)+C/P in Patients (pts) with BRCA1 or BRCA2 Mutations and Metastatic Breast Cancer: A Randomized, Phase 2 Study. 2016 San Antonio Breast Cancer Symposium, San Antonio, TX, 6-10 December 2016, Abstract S2-05.