Evidence Based Medicine, in Precision Oncology

The disagreements in clinical data and therapy recommendations extracted from different sources/studies are a common finding in oncology research. Knowingly “biology is less reproducible than physics and mechanic engineer-ing”, in order to overcome the disagreements and to find common grounds, we still rely on meta-analysis and systemic reviews for the highest level of evidence. To gather systemic review data base, a bibliographic search usually is conducted in the PubMed and in Cochrane Central Register of Controlled Trials databases to address a common clinical challenge. That said, frequently due to common conflicts between articles outcomes, an opinion of a third investigator is sought. Here in this article, we propose a rationale that could explain the differences in outcomes as a result of imperfect understanding of the current research database secondary to the unique biology of the tumor, rather than statistical interpretation on findings. We believe that the differences in findings merely are based on blinded inclusion criteria, and lack of accurate companion diagnostics to correlate the magnitude of response to each therapy. The objective of this article is to discuss a strategy to overcome such discordance by providing quantitative biological measures for genomic classification and correlation of tumor response to the selected targeted therapy. We further review such analysis in a case series of Her 2 positive breast cancer and conclude that translational research would be clinically relevant when customized to the biological findings. The Her 2 targeted was selected based on clinician’s and profile The proposed companion diagnostics (c were performed. We showed a wide range of laboratory findings. Presence of circulatory tumor cells and the expression of Her m RNA were quantified. Circulatory tumor DNA was assessed by Guardant 360 laboratory, and serum Her 2 level were checked through LabCorp, US. We reviewed the response to Her 2 therapy based on the laboratory findings. We able to successfully improve patients outcome by switching the therapy based on companion diagnostic dis-cussed.

unable to test targeted therapeutics against low frequency genomic "oncogenic driver" aberrations with adequate power. Usual accrual difficulties to clinical trials are exacerbated by low frequencies of any given molecular driver. To address these challenges, there is need for innovative clinical trial designs and strategies implementing novel diagnostic biomarker technologies to account for inter-patient molecular diversity and scarce tissue for analysis [1]. Current attempts in application of new targeted therapies for cancer have been based on the recent understanding of the tumor set of heterogenous colonies which each at certain point of time drives the tumor growth. Determination of these sets of colonies specific genomic alterations by biopsy (tissue and liquid) has provided extensive knowledge in exploring several actionable targets. That said, majority of the current drugs are approved based on the "presence" of a specific alteration rather than its quantitative measure of expression. For example, the presence of EGFR alteration in lung cancer, or Her 2/neu mutation, would prompt administration of EGFR targeted therapies. That said, the activated (mutated) target can "express" itself in a wide range, and more importantly its expression varies by time, and is not static [2] [3] [4]. Such dynamic effect has long been clinically underestimated, as the tumor initial response has been more of a focus than its duration of response. In fact, due to the tumor secondary mutations and changes in its expression of treated target, no one can really know how long a patient will respond to a specific targeted therapy. The amount of response also has only been estimated based on historical data in each study. For tumors that overexpress HER 2, trastuzumab-based chemotherapy is now the standard of care and it has improved the rate of response, time to progression, and the overall survival in patients with metastatic breast cancer, as well as disease-free and overall survival in patients with localized invasive breast cancer. That said, Her 2 positive breast cancer response to Herceptin, (if there is no inherent or acquired resistance reported) has a quantity and duration with a range of negligible to significant and between few weeks to years, depending of the clinical study performed and the candidates specific tumor character included in the studied.
Many of the studies that have looked at application of Gleevec for example in Her 2 positive breast cancer with hormonal blockade again disagree on the outcomes as the candidates were not stratified for the presence of TP53 or PDGF.
Such discordance and disagreements merely are caused by the lack of biological information on tumor behavior and its unique character in each case studied in the trial. As a result, next generation clinical trials are suggested to replace the current oncology clinical trial design to avoid the blinded conclusions in blinded trials.
Circulatory DNA and Circulatory tumor cells are accessible by a blood sample, and can provide significant information on tumor genomic profile in a dynamic fashion. Such information includes genomic presence and absence of mutations, amplifications and even m RNA findings at CTC level [5] [6]. With such information, it would be very feasible to assess the tumor's signature and

Method and Material
We  She reported improved quality of life after the very first treatment, and continued to improve clinically. Her ECOG improved 3 points from 3 to 1. Her labs were repeated and after two weeks on July 26th, and showed a marked reduction (20 percent) in all her tumor markers. Her c DNA completely resolved (please see Figure 1). Her calcium dropped to normal range at 8.    (Please see Figure 3).  Her CTC was repeated by biofocus on 12/17/15 and it showed complete eradication of CTC with CK20 and ERBB2. The C Myc virtually stayed the same.
On 1/20/2016, her CTC was repeated and it showed complete resolution of CTC post therapy. (Please see Figure 4). Her circulatory DNA was measured through Guardant and it was positive for PI3k and TP53, at high MAF, measured on 11/9/15, 12/1/15 and negative on 12/30/15 (please see Figure 5). She received Herceptin along with IV multitargeted epigenetic therapies, that could explain the disappearance of ERBB2 from her CTC and/or c DNA, however there is no expected positive effects from Herceptin on P53, or PI3K, nor the c MYC present at her initial CTC. We contribute such finding to the IV epigenetic therapies, she received at the interim, as this therapy in fact targets c MYC, P53 and Pi3k simultaneously. She stopped the IV epigenetic therapies on 1/16, as she moved back home, and stayed on Herceptin every three weeks, schedule, along with monthly Lupron and Xgeva, and tamoxifen. Her restaging scan in August 2016, showed significant progression of her disease with increased sizes and metabolic activities Journal of Cancer Therapy in all her lesions in thoracic and bones (New L 1 lesion with SUV activity of 16.9). Sternum enlarged tumor with SUV of 16.9 from 5.6, Sacrum lesion enlarged with SUV activity of 16.9 from 5.6, Right sacrum from 7.4 down to 3.7, Scapula from 5.2 to 13.1.
At this point the CTC was repeated and it showed the presence of HER 2 positive cells again. She immediately returned to our clinic and started the IV therapies again on 8/25/16, on daily basis. Her restaging scan was ordered on 9/15/16, and showed partial response to therapy with significant reduction in all her lesions sizes and SUV metabolic activities (cervical node, right breast implant 8.2 from 12.2, axillary nodes 4.2 from 6.0, , Pulmonary (hilar) nodules 4.3 from 9.2, and bony lesions in sternum 10.2 from 16, L1, from 16.3 down to 11.9. right scapula from 13.9 down to 8.4, sacrum from 7.4 to 6.6).
Her CTC again repeated in December 2016, showed complete resolution of the CTC post therapy (see Figure 6). This concept is novel as it could also substantiate our theory of targeted epigenetic therapies for driver epimutations in cancer, in this case could well correlate with the mutation identified at TET2 in the molecular profiling of this tumor. to Mexico and had right mastectomy. Her pathology showed ER/PR/HER 2 metastatic disease with bone involvement. She then was treated at St. Judes where she continued hormonal blockade, herceptin, zometa. She progressed slowly and was under the care of oncologist in Hanford. She also tried and failed Xeloda. She was started on Tamoxifen and offered radiation. Most recently she had been on Femara, but her most recent PET scan on 6/8/16 again showed progression of disease with complete destruction of the sterum. There were multiple lung mets, as well as extensive disease in the peritoneum. There were no liver lesions noted on her most recent PET. She has never had a paracentisis. She referred to us seeking alternative and integrative treatments. Upon her arrival she was evaluated and her labs drawn, which showed presence of CTC in the blood, along with expression of ERBB2 and c Myc. These CTC were ER negative (see Figure 7).
Her molecular profiling was performed which showed presence of several mutations in her tissue biopsied from her humerus. First they were Her 2 negative, second they were ER positive. Third they were MSH-6 positive making them responsive to the IV epigenetic therapies which started immediately on daily basis.
Her labs reported decreased tumor markers, CA 27.29 at 685 from 765, CA 15.3 at 533 from 574, Her 2 at 75 from 98, measured on 6/28/16 after two weeks of therapy. Her c DNA showed a marked reduction from 7.4 to 5.0 at the level of PI3k. Further as repeated it showed more reduction from 5 to 1.9, measured on 8/2/16 (please see Figure 8).
She received Herceptin after two weeks of MTET. Her initial response to MTET was reduction of her serum Her 2 level (please see Figure 9).
She did not respond to either Lapatinib or Herceptin, as her serum Her 2 increased again. This is a case of inherent resistance to Herceptin, and therefore she was started on epigenetic therapies again this time, with decreased serum Her 2. It was decided to use Afatinib (a pan EGFR blocker) in conjunction with Herceptin, in her case. Her tumor markers dropped and she continued to respond to therapy with improved markers, and continues to improve.

Conclusion
In all cases studied, there was a significant correlation between the mutational allele fraction (and presence) of c DNA, positive Her 2 CTC and serum Her 2 level with the magnitude of response. We believe that further hypothesis can be generated by this small sample looking at companion diagnostics of Her 2 disease before, during and after the therapy. It is also mentionable that there was some correlation between the serum Her 2 level and resistance to targeted therapy. We believe that the current standard of care should change in regards to initiation and the duration of Her 2 therapy, as relying on the progression of disease in scan, as an end point for the therapy could postpone sequencing or selecting a more desirable therapy. Furthermore these cases substantiate the evidence on effectiveness of multi targeted epigenetic therapies for driver epimutations in cancer.