Karanahan: A Potential New Treatment Option for Human Breast Cancer and Its Validation in a Clinical Setting.

INTRODUCTION: Karanahan, a cancer treatment technology aimed at eradicating tumor-initiating stem cells, has already proven effective in 7 tumor models. Karanahan comprises the following procedures: (1) collecting surgical specimens, (2) determining the duration of the DNA repair process in tumor cells exposed to a cross-linking cytostatic agent, and (3) determining the time point, when cells, including tumor-initiating stem cells, are synchronized in the certain phase of the cell cycle after triple exposure to the cytostatic, becoming vulnerable for the terminal treatment, which is supposed to completely eliminate the rest of survived tumor-initiating stem cells. Determining these basic tumor properties allows to design the schedule for the administration of a cross-linking cytostatic and a complex composite DNA preparation. Being conducted in accordance with the schedule designed, Karanahan results in the large-scale apoptosis of tumor cells with elimination of tumor-initiating stem cells. METHODS: Breast tumor specimens were obtained from patients, and basic tumor properties essential for conducting Karanahan therapy were determined. RESULTS: We report the first use of Karanahan in patients diagnosed with breast cancer. Technical details of handling surgical specimens for determining the essential Karanahan parameters (tumor volume, cell number, cell proliferation status, etc) have been worked out. The terminally ill patient, who was undergoing palliative treatment and whose tumor specimen matched the required criteria, received a complete course of Karanahan. CONCLUSIONS: The results of the treatment conducted indicate that Karanahan technology has a therapeutic potency and can be used as a breast cancer treatment option.

Association between Lymph Node Status and Expression Levels of Androgen Receptor, miR-185, miR-205, and miR-21 in Breast Cancer Subtypes.

Breast cancer is the most commonly diagnosed cancer among women. Difficulties in treating breast cancer are associated with the occurrence of metastases at early stages of disease, leading to its further progression. Recent studies have shown that changes in androgen receptor (AR) and microRNAs' expressions are associated with mammary gland carcinogenesis, in particular, with the formation of metastases. Thus, to identify novel metastatic markers, we evaluated the expression levels of AR; miR-185 and miR-205, both of which have been confirmed to target AR; and miR-21, transcription of which is regulated by AR, in breast cancer samples (n = 89). Here, we show that the molecular subtypes of breast cancer differ in the expression profiles of AR and AR-associated microRNAs. In addition, the expression of AR and these microRNAs may depend on the expression of PR, ER, and HER2 receptors. Our results show that the possibility of using AR and microRNAs as markers depends on the tumor subtype: a decrease in AR expression may be the marker for the presence of lymph node metastases in patients with HER2-positive subtypes of breast cancer, and disturbance of miR-205, miR-185, and miR-21 expressions may be the marker in patients with a luminal B HER2-positive subtype. Cases with metastases in this type of breast cancer are characterized by a higher level of miR-205 and a lower level of miR-185 and miR-21 in tumor tissues compared to nonmetastatic cases. A decrease in the miR-185 level is also associated with lymph node metastasis in luminal B HER2-negative breast cancer. Thus, the expression levels of AR, miR-185, miR-205, and miR-21 can serve as markers to predict cancer spread to the lymph node in luminal B- and HER2-positive subtypes of breast cancer.

Approbation of the Cancer Treatment Approach Based on the Eradication of TAMRA+ Cancer Stem Cells in a Model of Murine Cyclophosphamide Resistant Lymphosarcoma.

BACKGROUND/AIM: We previously have described the "3+1" tumors cure approach consisting of individual time schedule of cyclophosphamide and dsDNA preparation administrations. The aim of the study was to adapt the "3+1" approach based on eradication of cancer stem cells to the model of murine ascitic cyclophosphamide-resistant lymphosarcoma (RLS). MATERIALS AND METHODS: Adaptation of the "3+1" approach includes the identification of the timing to disrupt the tumorigenic potential of a certain tumor. RESULTS: The proposed therapeutic scheme allowed complete reduction of primary RLS ascites in experimental animals. However, reduction of primary ascites due to the complementary action of cyclophosphamide and dsDNA was inevitably followed by the development of a secondary one, most likely arising from a solid carcinomatous formation in the peritoneal wall. CONCLUSION: The "3+1" approach resulted in the elimination of cancer stem cells, and, as a consequence, in the complete reduction of RLS ascites.

Autologous dendritic cells and activated cytotoxic T­cells as combination therapy for breast cancer.

Breast cancer is the most common oncological pathology in women worldwide. Techniques for improving the clinical parameters of patients undergoing combination therapy for breast cancer are currently under development. A type of treatment employing dendritic cells (DCs) and cytotoxic DC­induced antigen­specific T lymphocytes efficiently eliminates residual cancer cells that are the key cause of tumor recurrence and metastasis. In the present study, DCs and activated lymphocytes (treated with IL­12 and IL­18) were isolated from the peripheral blood of patients with breast cancer, using a lysate of tumor tissue as antigen. The patients received the cells as part of adjuvant or neoadjuvant regimens (stage IV disease or progression). Evaluation of immunity was performed at 3 and 6 months after terminating immunotherapy. Evaluation of the disease­free period was performed for 3 years after surgery. The use of antigen­loaded autologous DCs combined with mononuclear cells with increased cytotoxic activity following Th1 polarization reduced the populations of immunosuppressive cells. The results of the present study demonstrated that the investigated cellular immunotherapy for breast cancer is safe, reduces the risk of relapse and metastasis, and improves immunity by reducing the number of regulatory T cells. Therefore, this therapeutic strategy may represent a novel approach to combating distant metastases of breast cancer.

Establishment of primary human breast cancer cell lines using "pulsed hypoxia" method and development of metastatic tumor model in immunodeficient mice.

BACKGROUND: Among breast cancer (BC) patients the outcomes of anticancer therapy vary dramatically due to the highly heterogeneous molecular characteristics of BC. Therefore, an extended panel of BC cell lines are required for in vitro and in vivo studies to find out new characteristic of carcinogenesis and metastasis. The purpose of this study was to develop patient-derived BC cell cultures and metastatic tumor models representing a tool for personal therapy and translational research. METHODS: Breast cancer cells were prepared by optimizing technique from tumor samples. We used real-time RT-PCR, flow cytometry, western blotting, cytotoxicity assay, karyotyping and fluorescent and electron microscopy analyses to characterize the established cell lines. BC xenografts in scid mice were used for in vivo tumorigenicity studies. RESULTS: The technique of preparing primary cells was optimized and this resulted in a high output of viable and active proliferated cells of nine patient-derived breast cancer cell lines and one breast non-malignant cell line. High E-cadherine and EpCAM expression correlated positively with epithelial phenotype while high expression of N-cadherine and Vimentin were shown in cells with mesenchymal phenotype. All mesenchymal-like cell lines were high HER3-positive-up to 90%. More interesting than that, is that two cell lines under specific culturing conditions (pulsed hypoxia and conditioned media) progressively transformed from mesenchymal to epithelial phenotypes displaying the expression of respective molecular markers proving that the mesenchymal-to-epithelial transition occurred. Becoming epithelial, these cells have lost HER3 and decreased HER2 membrane receptors. Three of the established epithelial cancer cell lines were tumorigenic in SCID mice and the generated tumors exhibited lobules-like structures. Ultrastructure analysis revealed low-differentiate phenotype of tumorigenic cell lines. These cells were in near-triploid range with multiple chromosome rearrangements. Tumorigenic BrCCh4e cells, originated from the patient of four-course chemotherapy, initiated metastasis when they were grafted subcutaneous with colonization of mediastinum lymph nodes. CONCLUSIONS: The developed BC cells metastasizing to mediastinum lymph nodes are a relevant model for downstream applications. Moreover, our findings demonstrate that pulsed hypoxia induces transformation of primary fibroblastoid breast cancer cells to epithelial-like cells and both of these cultures-induced and original-don't show tumor initiating capacity.

Characterization of primary normal and malignant breast cancer cell and their response to chemotherapy and immunostimulatory agents.

BACKGROUND: The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment. METHODS: Here, we developed primary cultures of human breast tissue - normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and ß, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized. RESULTS: We revealed that BC5 carcinoma cells were PGRlow/ERbhigh/ERa-/Cyp19+, the BrCCh1 cells that originated from the recurrent tumour were PGR-/ERb+/ERa-/Cyp19+, and normal BN cells were PGR-/ERb+/ERa-/Cyp19high. The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines. CONCLUSION: Our data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.

Five-year disease-free survival among stage II-IV breast cancer patients receiving FAC and AC chemotherapy in phase II clinical trials of Panagen.

BACKGROUND: We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response. METHODS: We analyzed 5-year disease-free survival of patients recruited into the trial. RESULTS: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %. CONCLUSIONS: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer.

BACKGROUND: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited. METHODS: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range. RESULTS: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort. CONCLUSIONS: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Effects of human exogenous DNA on production of perforin-containing CD8+ cytotoxic lymphocytes in laboratory setting and clinical practice.

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.

A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation.

BACKGROUND: Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth. METHODS: Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105 tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups. RESULTS: The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group. CONCLUSIONS: Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.