Researcher's recent intention to inhibit telomerase as a therapeutic measure has failed in general, as the cancer cells have a secondary adaptive cellular mechanism of resistance to such approach, using an "Alterative Lengthening Telomeres" (ALT) pathways, involving mitochondria, (by increased expression of PGC-1beta), to [1-7]. The highly aggressive and metabolically active cancer cells are able to survive regardless of the telomere's activity [8]. This has important clinical implications, as clinically we see cases that are treated with different therapeutic interventions and we can show that surprisingly, the telomerase activity increases with an effective therapy. This can be shown in the samples of the circulatory tumor cells that have no telomerase activity, but very highly aggressive features, such as G250 in renal cell carcinoma, which is suggestive of activated ALT [9,10]. After a successful therapy they lose their aggressiveness by a lack of G250, and they start to show positive telomerase activity in their signature in liquid biopsy. To date no clinical study or theory explains this. Here we present a case of renal cell carcinoma, which supports the concept and offers a possible explanation for presence of telomeres as a "sign of response" to therapy. We also present several case studies that show response to Multi-targeted Epigenetic Therapy, as evident by the eradication of telomerase positive cells in liquid biopsy.

Keywords: Cancer prevention, Telomeres, Epigenetic therapy
Case Studies
Sample Case 1: Renal cell carcinoma
67 year old male with a 10 cm renal mass incidentally found in an ultrasound, which was suspected for malignancy. He was scheduled for laparotomy, the biopsy was deemed to be too dangerous to perform. He was referred for evaluation and treatment on 8/11/14. Labs were drawn which showed increased VEGF at 248 (pg/ml), serum interleukin-8 of 448.4. His D-Dimer was also elevated at 0.61. He started the daily IV epigenetic therapies, consisting of Quercetin and Sodium phenyl butyrate as part of the patented multitargeted epigenetic therapy (MTET) protocol. He did not experience any negative side effects, and did not change his diet or his supplements.
His labs were repeated after 10 treatments (two weeks), which showed substantial improvements. His VEGF dropped from 248 down to 58 and his Interleukin-8 dropped from 448.4 down to 16.6. His CRP dropped from 160 to 133. He was scheduled for surgery, as his risk of angiogenesis has decreased significantly. His initial thrombocytosis as a sign of angiogenesis resolved, as his platelets dropped from 388 to 201. He had CTC analysis through Biofocus laboratory before and after the treatments. The results indicated significant reduction of the tumor burden in CTC as well as CTC markers, including CD20, telomerase, and cytokeratin markers. The results may indicate response in the micrometastatic environment. The markers for angiogenesis are suggested in our research to correlate with survival.
The first sample showed positive G250 and second has a negative G250. Telomerase overexpression was observed in the second sample, which suggests our concept with positive response to therapy, as explained above (Figures 1 and 2).
Sample Case 2: Metastatic malignant melanoma
29 year old female with history of melanoma, diagnosed in April 2012 status post biopsy of her right forearm nevus, side margin excision, and graft and LN biopsy. MRI showed disease free status, status post a recurrence in 9 months, with vision problem in January 2013, with multiple brain mets, including Chiasm of Optic nerve, status post 5 cycles of Immunophresis in Germany, Interferon, and Yervoy (5 mg/kg for two rounds), and subsequent Craniotomy by in Australia, and cyberknife of remaining lesion in brain. In February 2013, she had received Sutent. Her molecular profiling of her tumor showed positive NRAS mutations.
After initial evaluation, immediately she was started on IV epigenetic therapies which she received on daily basis. After ten treatments she expressed improvement in her function and vision. Her ECOG score improved as was her vision. Her labs were repeated which showed following results:
Neuron Specific Enolase (NSE) dropped from 45 to 9.2 and her interleukin 8 has dropped from 76 to 53, in two weeks, repeated on 4/28/14. LASA dropped from 64 to 27 (measured on 5/15/14 and 5/5/14 respectively).
We used Biofocus lab for her circulatory tumor analysis. The test was done PRE and POST epigenetic therapies. She did not receive any chemotherapies, or immune therapies or cytotoxic or targeted therapies during this time. Her CTC showed complete resolution post therapy, with disappearance of telomerase positive cells. Her CT scan was repeated on 6/2/14, which showed mixed response with many lesions in her chest and neck improved in size and activity.
This was a rapid response in an advanced case of RAS positive refractory to immune therapy in melanoma (Figures 3 and 4).
Sample Case 3: Squamous cell carcinoma of the lung
79 year old male with history of Stage IV squamous cell lung cancer, with metastasis to the shoulder, ribs, muscles, with some possible lung and peritoneal involvement. He was first diagnosed in April 2015, when he developed shoulder pain. An MRI done by orthopedic surgeon reveal lesion, and he was then referred to multiple doctors, resulting in a PET scan and referral to an oncologist.
He initially received Carboplatin and Abraxane with radiation in June 15, which failed. He was then switched to Nivolumab, which was given until his most recent scan in Jan 16 showed mixed results. Some of the radiated bone lesions responded, however metastatsis were still widespread. He lost over 25 pounds and had decreased quality of life after the therapies. He had not been given any other options other than palliative care.
His CTC done by Biofocus on 2/11/16 on showed positive EGFR overexpression and showed positive Telomerase.
He was started on IV epigenetic therapies immediately. Following 10 treatments, his CTC was repeated on 2/25/16, and showed eradication of the Telomerase positive cells, with no indications for remaining CTCs. His quality of life has also improved, as he has gained weight and his ECOG score improved from 2 to 1 (Figures 5 and 6).
Conclusion and Discussion
The literature has shown that in a majority of cancer cells, telomerase activity is present. As evident by the above cases studies, MTET therapy was able to suppress the telomerase activity. In the case renal cell carcinoma presented above, there was a paradoxical finding of increased telomerase as a marker of less aggressive cancer phenotype, due to accessory shifts. The clinical responses presented here, across various types of tumors, represents a potentially effective and novel method of treating cancers with metastatic disease, and needs further attention in large trials. Clinical investigations are required to evaluate the correlation of clinical response to presence of telomerase overexpression in the circulatory tumor cells.

1. Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene. 2002;21(4):598-610.
2. Blackburn EH. Telomerase and Cancer: Kirk A. Landon - AACR Prize for Basic Cancer Research Lecture. Mol Cancer Res. 2005;3(9):477-482.
3. Belair CD, Yeager TR, Lopez PM, Reznikoff CA. Telomerase activity: A biomarker of cell proliferation, not malignant transformation. Proc Natl Acad Sci U S A. 1997;94(25):13677-13682.
4. Blocking Telomerase Kills Cancer Cells but Provokes Resistance, Progression. Drug Week. 2012.
5. Xu T, Lu B, Tai YC, Goldkorn A. A Cancer Detection Platform Which Measures Telomerase Activity from Live Circulating Tumor Cells Captured on a Microfilter. Cancer Res. 2010;70(16): 6420-6426.
6. Flynn RL, Cox KE, Jeitany M, et al. Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors. Science. 2015;347(6219): 273-277.
7. Akincilar SC, Unal B, Tergaonkar V. Reactivation of telomerase in cancer. Cell Mol Life Sci. 2016.
8. Gunes C, Rudolph K. The Role of Telomeres in Stem Cells and Cancer. Cell, 2013;152(3):390-393.
9. Divgi CR, Pandit-Taskar N, Jungbluth AA, et al. Preoperative characterisation of clear-cell renal carcinoma using iodine-124-labelled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: A phase I trial. Lancet Oncol, 2007;8(4):304-310.
10. Cohen HT, Mcgovern FJ. Renal-Cell Carcinoma. N Engl J Med,2005;353(23):2477-2490.