Results of the feasibility study of clinical trials of the new cancer treatment technology Karanahan for patients with advanced breast cancer

Introduction

The challenges of palliative care of patients with advanced breast cancer

In modern oncology, there exist two fundamentally different cancer treatment options: the curative and the palliative ones (1-3). At non-advanced cancer stages, when there is a real chance that a patient will be completely cured, all therapy measures that aim to combat this pathological process are regarded as curative treatment options. Palliative treatment is used in advanced stages, when patients have virtually no chances for complete recovery (4-8).

Globally, there are more than 25 million cancer patients who belong to the palliative cohort; ~18% of these patients have breast cancer, ~12% colon cancer, and ~10% prostate cancer. The relationship between the number of diagnosed patients and those surviving for at least 5 years is a prognostic marker of overall survival (OS). Thus, this ratio is 3.8 for breast cancer; 2.7 for colon cancer; 1.5 for stomach cancer; and 1.0 for lung cancer (9).

Palliative chemotherapy is used with a deliberately nonradical objective for incurable patients with regional or distant spread of inoperable tumor processes. Meanwhile, palliative chemotherapy can increase the survival time of patients with types of spread cancers by several months or even years (3,8-15). It is conventionally assumed that when talking about solid chemosensitive tumors such as breast cancer, ovarian cancer, lung cancer, and colorectal cancer, the term ‚incurable‘ is used when distant metastases are present (stage IV) and, in some cases, when there is an inoperable regional metastatic process (stage IIIB for lung cancer) (16,17). Patients’ expected survival time plays almost no role in these cases. In patients with distant metastases, even if their functional status is satisfactory and the survival time can potentially be five years and even longer, the disease is considered incurable (18). It does not actually matter how long a patient will survive; what is important is that there are no chances of cure. In other words, if the probability that a patient eventually dies of current cancer is very high, the disease is considered incurable regardless of how long he or she stays alive (19).

Palliative chemotherapy aims to improve patient quality of life and survival time. However, it sometimes does not increase survival time compared to maintenance therapy and is highly dependent on the type and stage of cancer (20).

A typical feature of chemotherapy for patients with advanced cancer is that it does not involve a certain finite number of cycles. This therapy is given indefinitely as long as it helps curb the disease and does not cause serious adverse events. Tumor response to treatment is determined according to the RECIST guideline (version 1.1). Reduction in tumor size, attaining temporary remission, stabilization, or inhibition of the progression of the pathological process are sufficient to achieve the aforementioned objectives (18,21,22).

The challenge of decision making when performing palliative chemotherapy consists of ensuring the balance between quality of life and survival. In other words, the adverse symptoms of chemotherapy should not be a greater burden for the patient than the symptoms of the disease per se. Currently, there are no uniform standardized criteria for selecting cancer patients to receive palliative chemotherapy. For most tumors, details of the application of chemotherapy (indications to prescribe a particular antitumor agent or their combinations, administration procedure, and doses), rather than its appropriateness and effectiveness, are a matter of discussion. The key practical challenge of chemotherapy is associated with the details and methodology of its application (23). One of the well-known features of chemotherapy is that there are no unified generally accepted treatment protocols for all tumor types (13,18). There is even less certainty about palliative care for cancer patients, for which the objectives are quite different, as already mentioned. The age and poor general condition of patients with inoperable advanced cancer limits pharmacological therapy options and is often the reason why treatment is not performed at all (18). Therefore, the most challenging problem in palliative chemotherapy is choosing the optimal treatment strategy to solve the dilemma of how to conduct effective therapy while avoiding toxic effects. To have a high quality of life for patients, key clinical manifestations of metastatic cancer must be reduced, which is possible only through effective chemotherapy that in turn causes a number of unwanted adverse effects. In pursuit of the mandatory balance between the manifestations of the disease per se and the side symptoms of treatment, in most cases the attending oncologist/chemotherapist performs personified correction of therapy regimens, since no definite criteria or techniques for modifying palliative chemotherapy regimens are currently available (19).

Cyclophosphamide (CP) metronomic chemotherapy

CP is an alkylating chemotherapeutic agent that belongs to the oxazaphosphorine group. On exposure to cytochrome P450, it forms cytotoxic metabolites in the liver. CP is widely used in palliative therapy. In palliative chemotherapy using CP, the cytostatic is most effective when used in the low-dose metronomic regimen, or as monotherapeutic or in combination with other cytostatic agents. Metronomic chemotherapy is potentially a less toxic but quite effective treatment strategy, being a novel, active, and well-tolerated therapeutic modality for patients with advanced tumors (24).

Metronomic CP is a chemotherapeutic agent that is most commonly studied in preclinical experimental research (25,26). Thus, it has been demonstrated in animal models that metronomic administration of CP repolarizes M2-like tumor-associated macrophages toward the tumor-suppressive M1 phenotype, selectively reduces the number of circulating CD4⁺CD25⁺ regulatory T cells, while restoring the effector functions of T cells and natural killer cells, thus ensuring better control over the disease (27). Recent studies have demonstrated that metronomic chemotherapy using low-dose CP activates cytotoxic CD8⁺ T cells and can induce long-term immunological memory, manifested as rejection of the tumor grafted in GL261 mouse glioma models (28). In clinical practice, this approach has been approved for treating patients with metastatic breast cancer (29,30). It was demonstrated that therapy reduced the count of circulating regulatory T cells by 40% (P=0.002), while the count of tumor-specific effector T cells increased statistically significantly (P=0.03). This immunomodulatory effect was directly correlated with stabilization of the disease and OS of the patients (P<0.05). Metronomic chemotherapy has been included in the Updated Guidelines of the German expert group “AGO Breast Committee” where metronomic therapy is recommended for patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who had previously received anthracycline-taxane chemotherapy.

The chronometric Karanahan technology, the principles and therapeutic efficacy in the experiment, the position of National Comprehensive Cancer Network (NCCN)/European Society for Medical Oncology (ESMO) clinical practice guidelines

Multi-year research into the synergistic effect of CP cytostatic and complex composite double-stranded DNA preparation (DNAmix) has made it possible to develop the novel Karanahan technology for treating malignant tumors, which is based on the eradication of cancer stem cells. The Karanahan technology has been thoroughly described (31,32). This approach is of chronometric nature and implies the administration of both CP and DNAmix in strict dependence on the duration of DNA repair and the cell cycle pattern of each particular tumor. The introduction of the multi-component drug DNAmix at the demarcation point between the two phases of interstrand crosslink repair prevents the completion of nucleotide excision repair (NER) and initiation of homologous recombination. Prolonged stay in this “standby” state induces apoptosis, leading to the eradication of cancer stem cells. After being applied, the technology also induces extensive apoptosis of committed cancer cells and inhibits the suppressor activity of tumor-associated stroma. The therapeutic efficacy of the Karanahan cancer treatment technology is independent of both tumor immunogenicity and the patient’s immune status. While several elements of technology are also found in other therapeutic modalities, it is the novel therapeutic approach that uniquely integrates them to function concurrently and as a unified system, constituting the core mechanism of the treatment. The technology has been tested in different experimental murine tumor models: Krebs-2 ascites and solid carcinoma (33,34), hepatocellular carcinoma (G-29) with ascites (35), cyclophosphamide-resistant lymphosarcoma (RLS) lymphosarcoma (36), Lewis carcinoma (31), A20 lymphoma (37,38), as well as human tumor models: U87 glioblastoma (39), and primary glioblastoma cultures (40).

Low-dose metronomic chemotherapy with CP and Karanahan technology have the same chronometric platform for CP administration and therapeutic direction (41). Both treatment modalities reduce the pro-tumor activity of the tumor-associated stroma and activate the anti-tumor immune response. It is accompanied by complete regression of the nidus of immunogenic tumors in the case of low-dose metronomic chemotherapy with CP and nidi of primary tumors, both immunogenic and nonimmunogenic, in the case of using the Karanahan technology in experimental animals (37). Nonetheless, eradication of cancer stem cells rather than the antitumor immune response is the pivotal event in the Karanahan technology that predetermines curing of mice grafted with tumor of any genesis. Furthermore, the addition of DNAmix to therapy induces extensive apoptosis of committed cancer cells (33), thus reducing the tumor load and therefore increasing the overall therapeutic potential of treatment. This comparison gives grounds for suggesting that the Karanahan technology can be used as an option of palliative chemotherapy with CP having new therapeutic characteristics, namely the possibility of eliminating cancer stem cells from the tumor nidus and simultaneous induction of extensive apoptosis of tumor tissue cells. As it ensures stimulation of the antitumor immune response and reduces protumor activity of the tumor-associated stroma, this approach can be a potent therapeutic tool for patients with advanced cancer and stage IV breast cancer in particular.

Criteria to assess any novel therapeutic approach in breast cancer treatment have been developed in global practice (42-44). It is difficult to classify this approach under the existing clinical guidelines (NCCN/ESMO), since no data on clinically meaningful positive outcomes of the Karanahan therapy for breast cancer were available prior to the present study. Nevertheless, the substantial experimental evidence demonstrating significant therapeutic efficacy of the approach in treating incurable malignant neoplasms in mice and human glioblastoma xenografts may serve as a rationale for initiating human clinical trials of the therapy. We propose that the Karanahan technology can currently be classified as category of evidence 3 and category of preference 2/3 according to the NCCN Guidelines (45) and the case-control study level of evidence as defined by ESMO (46,47).

Analysis of the guidelines concerning the chronometric principles of the therapeutic approaches within the NCCN/ESMO system does not directly correlate with the chronometric principle underlying the Karanahan technology. The existing chronometric criteria take into account the cellular response to therapy, the need for immediate therapeutic intervention in aggressive breast cancer subtypes, the duration of administering therapeutic agents as long as a favorable therapeutic effect is observed, and the sequence of various therapeutic modalities. In the Karanahan technology, the chronometric regimen is determined by the tumor’s biological clock in response to the crosslinking cytostatic agent CP upon interstrand DNA crosslink repair and the exit of cancer stem cells from prolonged cell cycle arrest into a cell cycle phase susceptible to treatment. CP is employed not as a cytoreductive agent, but rather as a chemical reagent inducing interstrand crosslinks; inhibition of their repair constitutes the core principle of the Karanahan technology.

In the present study, the novel technology for treating malignant neoplasms, which had previously been tested in experimental animals, was used in a local clinical trial of the feasibility and practical applicability as a therapeutic procedure for the palliative treatment of advanced breast cancer. The major advantage of the novel therapeutic approach over existing breast cancer treatment regimens is that it has a targeted effect on cancer stem cells during cancer progression, when other therapies are unable to overcome the therapeutic resistance of the tumorigenic core. This characteristic, along with the simultaneous induction of apoptosis of committed tumor cells and disruption of the pro-tumorigenic activity of the tumor-associated stroma, gives grounds for expecting that this approach is effective for treating advanced breast cancer in palliative patients, provided that all the technological procedures in a clinical setting prove to be feasible. We present this article in accordance with the TREND reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2722/rc).

Methods

The MCF-7 cell culture

Cells were cultured in DMEM (Thermo Fisher Scientific, Cat# 41966029, Waltham, Massachusetts, USA) supplemented with 10% FBS (Capricorn Scientific, Cat# FBS-11A, Ebsdorfergrund, Germany) and 100 µg/mL gentamicin antibiotic. After triple exposure of cells to 1 µg/mL mitomycin C (Sigma-Aldrich, Cat# M4287-2MG, St. Louis, United States) once daily (days 3 through 10 after the first exposure to mitomycin C), a sample of MCF-7 cells was collected. Half of the cells were fixed in an equal volume of methanol to analyze the cell cycle distribution according to propidium iodide fluorescence and identify the cell synchronization time on the BD FACSAria III (Becton, Dickinson and Company, Franklin Lakes, USA). The remaining cells were incubated with 0.1 µg of TAMRA-labeled DNA probe in the dark at room temperature; then they were washed and deposited onto slides by cytospinning, coated with 4',6-diamidino-2-phenylindole (DAPI)/1,4-diazabicyclo[2.2.2]octane (DABCO) under the coverslip; the count of TAMRA⁺ cells was evaluated on a Zeiss Axio Imager M2 fluorescence microscope (Carl Zeiss Microscopy, Oberkochen, Deutschland) (48).

DNAmix

DNAmix is a combination of three pharmacopoeial substances included in the Register of Pharmacologic Substances of the Russian Federation, which are to be mixed strictly as follows: (I) the preparation of fragmented salmon sperm (Derinat) (Registration Certificate No. 002916/01 dated February 27, 2008); (II) chlormethine, a crosslinking cytostatic drug of direct action (CAS: 51-75-2); and (III) the preparation of fragmented human DNA (Panagen) (Registration Certificate No. LSR-004429/08 dated June 9, 2008). The size of the DNA fragments is 200–4,000 bp. DNAmix is the subject of industrial property of KARANAHAN LLC.

Choosing the clinical study strategy

Any large-scale clinical study starts with assessment of its viability in a certain country. The study of feasibility and practical applicability is the earliest evaluative tool in this process, providing a definitive answer: either “yes, it is feasible” or “no, conceptual revision is needed”.

The study of feasibility and practical applicability, which is distinct from a pilot study, does not necessarily replicate the full structure (i.e., the complete protocol) of a future definitive clinical trial. Its objective is to evaluate various individual aspects of the protocol of the subsequent large-scale trials. Furthermore, the study of feasibility and practical applicability allows for design optimization, which may include refining the inclusion/non-inclusion criteria, choosing the optimal dose and adjusting temporal parameters. The development or adjustment of elements of the intervention procedure based on preliminary experimental data can also be performed. Beyond the analysis of protocol elements, the studies of feasibility of the project address “infrastructural” issues. These include assessment whether clinical sites are ready to conduct research and whether there is a sufficient number of patients who can be enrolled within a given timeframe; calculating the patient consent rate; and evaluating costs and ethical barriers. For local branches of pharmaceutical companies and contract research organizations within a specific country, it constitutes a preliminary stage prior to trial placement and significantly affects the decision regarding trial conduct (49,50).

Therefore, the primary objective of the present clinical trial of the Karanahan technology was to determine the feasibility and practical applicability of the project aiming to gain a clear understanding of whether further studies can be conducted as standard clinical trials. An additional objective of the project was to develop and obtain approval of the clinical trial protocol for evaluating and refining several individual aspects of future pilot and large-scale randomized trials, namely: patient enrollment; design optimization, including the adjustment of inclusion/non-inclusion criteria; technical aspects of the intervention procedure (determining the required volume of tumor material, confirming the presence of dividing tumor cells, choosing the optimal dose and preparation administration topology; as well as modifying temporal parameters).

Clinical study

A clinical study of personalized chemotherapy for breast cancer with low doses of CP was conducted (ClinicalTrials.gov ID NCT06361264, https://www.clinicaltrials.gov/study/NCT06361264). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Local Ethics Committee of the Research Institute of Fundamental and Clinical Immunology (protocol No. 120 of November 07, 2019). Written informed consent to participate in the study was obtained from each of the patients, which specified open publication of the results presented as reports or otherwise.

The objective of the clinical trial

To assess the feasibility and practical applicability of the Karanahan technology in clinical practice for treating advanced breast cancer. To transfer the technology from the laboratory protocol to a clinical setting. To additionally refine the intervention procedure. To evaluate the safety and clinical efficacy of the Karanahan technology in treating breast cancer (stage IV or disease progression).

Clinical outcome assessment (efficacy and safety)

• Clinical assessment will be conducted prior to treatment initiation, one month following the second therapeutic procedure, as well as 9 and 18 months after treatment initiation (examination of patients by their attending physician weekly throughout each therapeutic procedure and subsequently once monthly during the entire follow-up period).
• Patients will be followed up during 36 months after treatment initiation.
• The overall clinical examination (performed at baseline, one month after the second therapeutic procedure, as well as 9 and 18 months after treatment initiation) will include a complete blood count, urinalysis, blood chemistry test, and magnetic resonance imaging (MRI) of the organs.
• Quality of life will be evaluated using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) questionnaire specifically adapted for cancer patients.
• Clinical efficacy will be assessed using the World Health Organization (WHO)-recommended criteria for therapy response evaluation according to the RECIST 1.1 guidelines and by analyzing the patients’ survival time.
• The response of the primary tumor site to therapy and the survival time of palliative patients were regarded as the primary efficacy endpoint for the Karanahan technology.
• The assessment of the immunomodulatory effect will involve determining the peripheral blood levels of CD4+CD25+FoxP3+ regulatory T cells and CD8+CD107a+ T cells before and after chemotherapy (upon completion of the second treatment cycle).

Primary endpoint

Safety assessment involving the analysis of adverse reactions (both local and systemic) monitored weekly after treatment initiation.

Secondary endpoint

Assessment of clinical efficacy (according to the quality of life criteria, relapse-free and OS); assessment of immunomodulatory effects.

Inclusion criteria

• Stage IV breast cancer or progression of the disease with the presence of foci accessible for biopsy of tumor material;
• Complete awareness of the patient about the prognosis of the disease and the proposed treatment;
• The volume of tumor material required for vital assessment of the time parameters of the individual reparative cycle of tumor cells must be at least 4 cm³;
• Tumor cells transferred to primary culture must be in a state of proliferative activity.

Exclusion criteria

• Severe decompensated cardiovascular, respiratory, hepatic, renal failure;
• Presence of an acute infectious disease;
• Intolerance to CP;
• Severe neutropenia—the content of neutrophils is less than 1,000 per 1 µL of blood;
• Simultaneous participation in another clinical trial.

Patients

Patient information is summarized in Table 1.

Intervention details

Patients had stage IV breast cancer or disease progression with the presence of foci accessible for biopsy of tumor material. All the intervention elements (administration of the cytostatic agent and the DNAmix preparation, collection of resected material, and sampling blood for analysis) were explicitly included in the informed consent form, which was signed by each patient.

Tumor samples were handled according to the procedure described (32). Primary cultures were obtained; TAMRA⁺ cells were counted. The duration of the DNA repair process and the time of cell synchronization after three exposures to mitomycin C were determined. The CP administration schedule was determined for each patient according to the resulting time points.

According to the elaborated regimen, patients received 4 intravenous CP (Baxter Oncology GmbH, Endoxane, Halle, Germany) injections at the dose of 300 mg/m² in combination with 4 injections of 1–12 mg of DNAmix (KARANAHAN LLC) (51) administered to prominent tumor nidi and lymph depots. The patients received 2 to 6 courses of therapy. The interval between courses was 21 days.

Comparator group

A historical control group was used for ethical reasons. This cohort consisted of patients with a palliative status receiving treatment at the same clinic, using the same therapeutic protocols adopted in the Russian Federation, who presented with comparable tumor characteristics and were undergoing standard treatment (Table S1).

An obligatory comment for schedule determination

Calculating the repair cycle time starts at the point where the number of double-strand breaks is minimal. If this value is minimal at the zero point, the calculation starts at the zero point. The reason is that active transcription occurs in tumor cells, giving rise to transient double-strand breaks that are detected by the comet assay at the zero point. It is the minimal number of double-strand breaks after transcriptional pausing that means that the transcriptional activity of the cell has been paused and double-strand repair (i.e., the DNA repair cycle) has started. Therefore, the first time interval of the repair cycle involves all the time until the second minimal number of double-strand breaks. The time interval of the true DNA repair cycle starts with the first minimal number of double strand breaks and ends with the second minimum. This interval is included in the schedule after the first treatment is extended by the time it takes for transcriptional activity to end.

Why does triple exposure to CP lead to cell cycle synchronization of cancer stem cells? The repair time for different chromosome regions is related to the presence of repetitive DNA in them. The repair dynamics looks as follows. Double-strand breaks in euchromatin are repaired first, followed by repair of double-strand breaks in intercalary heterochromatin and possibly telomeres; finally, the repair machinery fixes double-strand breaks in centromeric heterochromatin. The multiple number of sites to be repaired, in combination with spatial hindrance to the molecular repair machinery in centromeric heterochromatin, are the possible reasons for the synchronization of the DNA repair machinery in different cells. At the initial repair stage, all easily accessible sites bind to the DNA repair complex and are fixed. Centromeric heterochromatin remains a region that is difficult to access for repair; as suggested earlier, the kinetics of binding of the repair machinery to sites within this region is low. In other words, all the energy was originally put into the repair of easily accessible sites. Once DNA repair at easily accessible sites is completed, all the DNA repair complexes simultaneously reach the sites in centromeric heterochromatin. The number of these complexes apparently is equal to or greater than the number of sites to be repaired. All the breaches are closed synchronously. This situation is obviously dependent on the cytostatic doses and the number of interstrand crosslinks induced by them.

Assessment of regulatory T cells and CD8 T-cell population in patients’ peripheral blood

Peripheral blood mononuclear cells were isolated by centrifugation of whole heparinized venous blood from patients in a density gradient of Ficoll-Paque^TM PLUS solution, ρ=1.077 g/mL (PanEco, Moscow, Russia).

Regulatory T cells (CD4⁺CD25⁺FOXP3⁺) were quantified by flow cytometry on a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, USA) using anti-CD4 (FITC), anti-CD25 (PE-Cy7), and anti-FOXP3 (PerCP/Cy 5.5) monoclonal antibodies (BD PharMingen, San Diego, CA, USA).

Externalization of CD107a on the surface of CD8 T cells was assessed using a degranulation assay. Mononuclear cells were incubated in the presence of anti-CD107a antibodies, monensin, and anti-CD3 antibodies as a T-cell stimulator. Anti-CD3 antibodies were not added to the control. After 18 hours, cells were incubated with anti-CD8 antibodies according to the standard procedure for detecting cell surface antigens. The level of CD107a expression was determined on a flow cytometer at the CD8 cell gate.

Statistical analysis

Statistical analysis was performed using Statistica 8 software (StatSoft, Tulsa, USA). The validity of the differences was evaluated using the two-tailed Mann-Whitney U test that is routinely used for small samples. The differences revealed were considered statistically significant at P<0.05.

Results

The study involved three stages. In the first stage, we analyzed the effect of the Karanahan technology on MCF-7 cell culture as a common breast cancer model. This stage was needed to prove the fundamental efficacy of breast cancer therapy in a cell model. The feasibility and practical applicability of the novel Karanahan technology in palliative patients with incurable metastatic breast cancer were assessed at the second stage. The third part of the study of reasonability and practical applicability involved additional evaluation and refinement of certain intervention procedures under the protocol of Karanahan therapy in palliative patients with incurable metastatic breast cancer, which is necessary to properly plan the design of randomized clinical trials.

The effect of the Karanahan technology on changes in the size of cancer stem cell population and viability of MCF-7 breast cancer cells

Diagnostic analysis of the Karanahan technology involves evaluation of the cellular repair cycle and distribution of cells by phases of the cell cycle after exposure to a crosslinking cytostatic agent in accordance with the identified time parameters of the repair cycle (Figure 1). To analyze the DNA repair cycle in MCF-7 cell culture, cells were treated with the direct-acting crosslinking cytostatic agent mitomycin C. After the maximum accumulation of double-strand breaks had been found (Figure 1A), cells were exposed to the mitomycin C cytostatic agent three times in a 22-hour interval (Figure 1B). The cell distribution in the phases of the cell cycle was analyzed within 10 days after the first cytostatic treatment. The final treatment point for MCF-7 cells (according to the Karanahan technology procedure) was chosen to be 6 days after the first cytostatic treatment (Figure 1C).

Figure 1 Diagnostic analysis of the Karanahan technology for MCF-7 breast cancer cells. (A) The DNA repair cycle in MCF-7 cell culture. (B) Scheme of treating MCF-7 cells with mitomycin C. (C) The cell cycle of MCF-7 cells after triple treatment with mitomycin C. Black arrows indicate the selected time for drug injections. (D) The percentage of TAMRA+ cancer stem cells in MCF-7 cell culture after triple treatment with mitomycin C. PI, propidium iodide.

Efficacy against cancer stem cells is one of the key parameters that characterize the Karanahan technology. In earlier study (32), it was demonstrated that TAMRA⁺ breast cancer cells substantially overlap with conventional breast cancer markers and are cancer stem cells in patients with this disease. Hence, we quantified changes in the percentage of TAMRA⁺ cells and therefore MCF-7 cancer stem cells throughout treatments (Figure 1D). The percentage of cells was found to reach the minimal value on day 6 and then increase abruptly. This value is as expected, since massive apoptosis of committed cells is induced at this time, and the relative percentage of apoptosis-resistant cells (cancer stem cells being among them) will obviously increase. Furthermore, once cancer stem cells complete repair, they proceed to division, which also contributes to the increase in the percentage of TAMRA⁺ cells.

DNAmix, which is the cornerstone of the Karanahan technology, was not supposed to be added to the therapy protocol. The effect of DNAmix was repeatedly evaluated in similar experiments for cancer cell cultures and did not require mandatory validation.

The findings demonstrated that breast cancer cells, both committed and TAMRA⁺ cancer stem cells, were sensitive to the Karanahan technology and that this technology can be applied in clinical practice under experimental conditions for incurable palliative patients who have the terminal stage of the disease.

Assessment of the feasibility and practical applicability of the Karanahan technology in clinical practice

The conducted procedures attested to the feasibility and technical viability of conducting clinical trials of any scale. Specific provisions requiring consideration when planning of clinical studies were identified. The necessary and sufficient volume of a resected specimen needed to use the technology was found to be 4 cm³. This volume is always attainable for patients with advanced breast cancer being the inclusion criteria of the study. The rate of patient accrual into the protocol indicated that large cancer treatment centers of federal level characterized by high patient throughput need to be involved for planning large-scale clinical trials. The laboratory analysis presupposes that a well-equipped laboratory with a team of personnel proficient in cellular and molecular biology is available. Furthermore, the legal aspects pertaining to the feasibility of conducting clinical trials in the Russian Federation were analyzed. It was ascertained that the subject matter of clinical trials will be the personalized schedule of agent administration.

The results of clinical trials

Certain individual aspects of the protocol of future large-scale trials (testing the components of the intervention procedure) were evaluated in the third part of the study. Study design was optimized (with the inclusion/non-inclusion criteria adjusted) and the technical elements of the technology were modified (determining the requisite volume of resected tumor material, confirming the presence of dividing tumor cells), finding the optimal dose and topology of agent administration, as well as choosing the appropriate temporal parameters for agent administration. A clinical study protocol was developed and approved by the Academic Council and the local Ethics Committee for this part of the study.

The general methodology of the Karanahan technology is as follows (32). During the preparatory stage, a tumor sample is harvested intraoperatively. The primary culture is obtained from this tumor tissue sample. The number of cancer stem cells is quantified according internalization of the TAMRA⁺ DNA probe. The repair cycle time is then estimated, and the day the tumor cells are synchronized in the G2/M cell cycle phase is identified. The schedules for administering CP and DNAmix are calculated according to the resulting time points. The schedule is handed to the physician in charge and therapy is performed. According to the elaborated regimen, CP is administered intravenously, while DNAmix is injected into prominent tumor nidi (Figure 2A). To increase bioavailability of the preparation, a decision was made during the trial to inject two additional doses of DNAmix of into lymph node depot areas in inguinal and axillary lymph node clusters and the abdominal lymph node 5 cm to the right of the umbilicus (paraumbilically) (Figure 2B).

Figure 2 Injections of DNAmix. (A) The procedure for intratumoral injection of DNA preparation; (B) additional zones for administering the DNAmix preparation according to the Karanahan technology. DNAmix, complex composite double-stranded DNA preparation.

The objective of the present clinical trial was not complete curation of stage IV breast cancer patients like it was for the experimental mouse models. The primary objective of the study was to test the applicability of the Karanahan technology in humans in a clinical setting. We needed to understand whether the technology works in a clinical setting, what drawbacks it has, and what are the hidden pitfalls for this therapeutic approach. In this regard, the key criterion for assessing the efficacy of the Karanahan technology was the response of the primary tumor nidus to therapy and the survival time of palliative patients.

Results of the Karanahan therapy in patients

At the time when the results were reported, 12 palliative patients diagnosed with stage IV breast cancer had participated in the study. The patients received two to six therapy courses using the Karanahan technology.

Safety of the proposed therapeutic approach was a critical consideration throughout this study. The total dose of the cytostatic agent CP administered during one treatment cycle was 1,200 mg/m² within 21 days, which is below the single therapeutic dose of CP for which a moderate toxicity and good tolerability has been demonstrated as reflected in the widely used standard CP-based regimens [e.g., FAC (fluorouracil, adriamycin, cyclophosphamide) and AC (adriamycin, cyclophosphamide)]. For the DNAmix preparation, long-term studies using murine models, along with preclinical and clinical studies of Panagen, a human double-stranded DNA (dsDNA)-based drug, revealed no toxic effects for the administered therapeutic doses. Therefore, the primary question was related to understanding the potential toxicity of the overall procedure. The Karanahan technology induces necrotic destruction of tumor tissue, which is accompanied by the release of a substantial mass of tumor debris into the systemic circulation. In murine models, this process in some cases elicited a systemic inflammatory response, leading to multiple organ failure and death of animals. It was fair to expect that a similar phenomenon, with varying severity, could occur in a clinical setting. For a certain indeterminate amount of this necrotic debris and the unpredictable duration of this process, grade III or IV adverse events can potentially occur. In this connection, continuous monitoring for the potential onset of a systemic inflammatory response was needed to control safe application of the Karanahan technology. It was achieved through patient examinations at key time points, by conducting comprehensive clinical tests, including complete blood count, urinalysis, and blood chemistry panels, and assessment of the emergence of adverse events. No grade III or IV adverse events having a prominent effect on patient’s life or being life-threatening were recorded.

To improve the monitoring of systemic inflammatory response development, future research protocols will comprise analyzing C-reactive protein concentration during Karanahan therapy as the key marker for incipient inflammation at the following time points: pre-therapy (day 0) and on days 14 and 21 following therapy initiation. A 14-day course of compensatory therapy aiming to facilitate the clearance of circulating debris from the bloodstream must be administered if there emerge signs of an incipient inflammatory process.

The compensatory therapy scheme for preventing the development of a systemic inflammatory response:

• Prevention of capillary microthrombosis—
  • Clexane^®, low-molecular-weight heparin, 0.4 mL syringe once daily, subcutaneous injections into the abdomen.
  • S-NaCl 0.9% 1,000 mL intravenously.
• Kidney protection, prevention of crystal formation in renal tubules and washing them—
  • Torasemide, 1/2 tablet twice daily.
  • S-NaHCO₃ 4% 200 mL intravenously.

Several patients were withdrawn from the study because of treatment-induced active catabolic processes (active necrosis with massive rejection; severe hyperemia) in the tumor subjected to therapy. The detoxification procedure was developed and used starting with the fourth Karanahan therapy course in patient 12.

The results are individually reported for the cases included in the study (supplementary material available at https://cdn.amegroups.cn/static/public/tcr-2025-1-2722-1.pdf). Data describing the occurring changes are collected and summarized in tables. Since all patients had metastases of different localization and the effect of treatment could refer to all affected areas, the efficacy of treatment was evaluated with account for the response observed in all nidi affections. Three response categories were selected: favorable response, no response, and unfavorable response. Tables list descriptions of changes according to response categories with allowance for all nidi of tumor dissemination according to the results of the specified instrumental analyzes on specified dates after treatment. The figures involve the data from laboratory examination of the Karanahan technology indicators and provide an illustration of changes that occur within the primary tumor site (photographs) during treatment.

The results for patient 1 were used for analysis twice and labeled entries Nos. 1 and 1-2. That was because after two courses of treatment using the Karanahan technology, tumor nidi in patient 1 was completely eliminated and no signs of tumor growth were observed for 2.5 years. However, after 3 years, this patient was readmitted for treatment, labeled entry No. 1-2. Tumor cell parameters were again evaluated and therapy was performed with the new regimen. We found it reasonable to analyze these two cases separately.

The following amendments were made and additional analyzes were performed throughout the study.

Patient 9

After the third course of therapy, biopsy material from the tumor area was analyzed to determine whether tumor cells were present; the schedule was supposed to then be adjusted. The tumor stroma sample did not contain a noticeable amount of tumor cells or any other cells. Of course, no trace of TAMRA⁺ cancer stem cells was detected. This fact, like in the case of experimental mouse models, indicates that treatment using the Karanahan technology is accompanied by fundamental positive changes in the tumor-associated stroma of the primary nidus. Tumor cells, including cancer stem cells, disappear, and only fibrous tissue remains.

Patient 12

The biological status of the cell populations in the pleural fluid was evaluated after three therapy courses. Cells in carcinoma nidus were found to have acquired a clear timing of DNA repair with a single dominant peak. The schedule was adjusted to account for the new findings. Furthermore, the dose of DNAmix administered increased from 1 to 12 mg per treatment session. No adverse events related to the injection of an escalated DNAmix dose were documented. Finally, in order to increase the chances of delivering DNA to the target metastatic nidus in the liver, peritoneum, and thoracic lymph nodes, we started to inject DNAmix into lymph depot areas in inguinal and axillary lymph node clusters, the abdominal lymph depot 5 cm to the right of the umbilicus (paraumbilically), and into prominent tumor nidi (Figure 2B). After adjustment of the procedure, the pleural effusion decreased in both terms of volume and frequency of evacuation (30 vs. 2–4 days between evacuations).

Hence, the following modifications to the protocol of therapy using the Karanahan technology were made for further clinical trials.

• The dose of DNAmix preparation should be escalated from 1 to ≥12 mg.
• The preparation DNAmix should be administered not only intratumorally but also into the lymph depots (into inguinal lymph node clusters, axillary lymph nodes, and paraumbilically) (Figure 2B).
• The C-reactive protein level must be analyzed during the Karanahan therapy. If signs indicating the onset of a systemic inflammatory response appear, detoxification measures must be initiated to prevent its progression.

Pilot assessment of treatment efficacy was also conducted. Table 2 summarizes the general conclusions about the results of Karanahan therapy for all patients.

Assessment of the survival time for non-survivors in the experimental cohort vs. historical control

The survival time of patients after palliative status has been given is one of the socially significant criteria in evaluating the efficacy of treating patients with conditions selected to conduct the present study. The evaluation of this parameter is presented in Table S1. The parameters of the experimental group and the historical control are compared.

A comparative analysis of survival time demonstrates that the Karanahan technology significantly increases the time interval before patient death (Figure 3).

Figure 3 Comparative analysis of the survival time of patients in the control group and in the experimental group (receiving therapy using the Karanahan technology). *, statistically significant differences, P<0.05, Mann-Whitney U test.

Quality of life assessment

Patients’ feedback on quality of life and tolerability of treatment was collected throughout the study. The resulting data are summarized in Table 3.

The quality of life score was significantly increased for three patients (Nos. 9, 10, and 11), being consistent with the objective indicators after therapy using the Karanahan technology. A significant worsening of well-being was observed for a single patient (No. 6), which was consistent with the fact that local tumor progression was detected and new metastatic foci emerged in this patient.

Tumor response according to the RECIST system

The clinically manifested tumor response observed after therapy with the Karanahan technology was analyzed. The results of the analysis were brought in line with the criteria of RECIST 1.1 and are summarized in Table 4.

Complete or partial tumor response to the Karanahan technology therapy or disease stabilization was observed in eight patients; disease progression occurred in two patients (Nos. 6 and 7). Another two patients (Nos. 2 and 4) had a positive local response, but disease progression (emergence of new metastatic foci) was also observed. Despite disease progression in patients 6 and 7, their OS time was substantially longer than the mean value in the historical control group (10 and 34 months, respectively). This outcome can be attributed to the incomplete, yet therapeutically positive, effect of Karanahan therapy. We hypothesize that the development of an intravenous DNAmix formulation would fundamentally enhance the efficiency of drug delivery to disseminated cancer stem cells and distant metastases, thereby significantly improving the efficacy of their eradication. Experimental work to investigate this hypothesis is currently underway.

Quantification of regulatory T cells and CD8 populations in peripheral blood

Determining the response of the immune system to the conducted therapy is a required analysis. We selected two parameters that provide a general view of the antitumor immunity level (Figure 4). An analysis of changes in the number of regulatory T cells allows one to draw a conclusion about the self-defense potential of the tumor. An analysis of changes in the number and cytotoxic activity of cytotoxic T cells gives grounds for drawing a conclusion about antitumor immune responses.

Figure 4 Quantification of regulatory T cells and CD8 populations in peripheral blood. (A) Content of CD4/25/FoxP3 cells in patients’ peripheral blood before and after Karanahan therapy. (B) Results of degranulation test with lymphocytes from patients’ peripheral blood before and after Karanahan therapy. “0” are the values before treatment. (C) The percentages of patients who had an increased, the same or reduced number of CD4/25/FoxP3 cells and degranulation test values are presented.

The degranulation of cytotoxic lymphocytes is the key indicator of the maturity and cytolytic activity of T cells, which is associated, among other factors, with their effect on tumor cells through specific antigens. Lysosomal granules containing cytolytic proteins are present in T cells. In differentiated CD8⁺ T cells, TLR-mediated stimulation causes degranulation (i.e., release of the contents of the lytic granules). Meanwhile, a CD107a molecule, a component of lytic granules, is placed on the surface of the T cell, which is used as a functional marker of cytotoxic lymphocytes. The externalization of CD107a on the surface of CD8 T cells was evaluated in the degranulation test.

The conducted analysis shows that the suppressive effect of the tumor on the body after therapy with the Karanahan technology is not fundamentally changed, while the activity of the adaptive antitumor immune response increases significantly.

Discussion

A universal marker of cancer stem cells and its application in the mechanism of eradicating cancer stem cells in breast cancer

In our studies conducted over the past five years, we have discovered and performed cell-level characterization of a general biological phenomenon that has remained unknown until recently: the ability of poorly differentiated cells, including cancer stem cells, to internalize fragments of double-stranded extracellular DNA via a natural internalization mechanism without employing the transfection procedure. A unique universal marker of stem cells of different genesis, including cancer stem cells, was fabricated using a fluorochrome-labeled DNA probe.

It was found simultaneously that double-stranded DNA fragments internalized in the cellular space of cancer stem cells during repair of CP-induced interstrand crosslinks interfere with the DNA repair process in such a way that they completely eradicate the malignant potential of the grafted tumor. A tentative mechanism of this phenomenon is likely to be mediated by several molecular processes. Extracellular DNA, present within the cell during activated DNA repair, may become directly involved in these pathways (52). During the initial repair phase (NER), interference with NER related to sequestration of repair proteins by fragments of native human double-stranded DNA is the most plausible mechanism. A similar mechanism has been well-documented in academic literature. Furthermore, a novel trend in cancer treatment has recently emerged: nucleic acid (NA)-based cancer molecular therapies. Short synthetic oligonucleotide duplexes [small interfering DNA (siDNA)] are used in this case: when delivered into a tumor cell, they mimic natural double-strand breaks, thus engaging DNA repair enzymes and disrupting the proper course of the repair process (53). The crosslinked and non-crosslinked dsDNA from fish, the components of the DNAmix therapeutic preparation, interfere with the second repair phase proceeding via the conservative homologous recombination mechanism. It is hypothesized that these foreign modified dsDNA fragments dynamically compete for binding to the 3 filament of the lagging strand at the site of replication fork restoration for a long time. The prolonged persistence or erroneous resolution of the intermediates formed during both repair phases may cause lethal consequences for tumorigenic cells (54). In both scenarios, the aberrant completion of repair phases prevents cancer stem cells from cell cycle re-entry, ultimately leading to apoptotic cell death.

The elimination of cancer stem cells from the mass of engrafted cancer cells was the key factor in the loss of tumorigenic properties by the graft (48,55-57).

Therefore, we have experimentally identified two components that underlie a fundamentally new strategy to treat malignant neoplasms. Furthermore, the general biological nature of the discovery allows us to focus on novel ways to influence stem cells and develop novel technologies for manipulating cellular genetics and properties.

Therapeutic effect of the Karanahan technology

In this pilot project, we have challenged ourselves to evaluate the potential favorable effects of the novel technology in real clinical practice for the breast cancer model in cases of stage IV cancer. Breast cancer was chosen as a model disease due to its high prevalence as well as the social significance of successful treatment of this disease. Being fully aware of the legal and procedural challenges associated with the novel therapy, we focused on palliative patients with advanced stage IV breast cancer. The possibility of observing favorable clinical effects in such advanced cancer cases could justify the search for clinical regimens that allow the elimination of tumor nidus and bring patients to remission even at such an advanced stage of the disease.

An analysis of the effect of the Karanahan technology on stage IV breast cancer progression revealed that (I) the survival time for analyzed patients was significantly increased, thus undoubtedly being a socially significant outcome, and (II) the positive dynamics within the most pronounced tumor nidi was observed locally, at the DNAmix injection sites.

Patient 2 had tumor nidi on both breasts. The tumor in the right breast was palpable; the tumor in the left breast occupied the entire gland volume and was a very dense conglomerate. After two cycles of therapy, the tumor in the right breast was no longer palpable. The affected tissue was rejected as necrotic fistulas. The tumor in the left breast became significantly smaller and soft. In other words, the primary nidus had undergone a positive transformation.

In the case of patient 9, after three consecutive treatments using the new technology, the necrotizing tumor shrank and became smaller; tissue decay almost stopped; the edges of the wounds of the surface were epithelialized; and the ichorous odor subsided. Tumor cells could not be isolated from the tissue sample after three therapy cycles, and the nidus appeared to have transformed into a fibrous stroma.

As mentioned above, although the patients’ survival time had increased significantly, a pronounced systemic response and a reduction in the size of all tumor nidi in the liver, bones, and skin were not achieved in a particular situation. We believe that the insufficiently pronounced systemic response is caused by two factors. First, it is the inadequate estimation of the biological clock of the DNA repair cycle and the cell cycle of tumor cells, which is related to the state of the original cellular material and will be difficult to correct. Second, there is the problem of delivering the required drug doses to the tumor nidus at the right time and the loss of effective dose associated with it. Thus, phosphoramide mustard, the CP metabolite that is formed in the liver, either does not reach the target zones due to poor blood supply (for skin and bone metastases) or is broken down as it passes through systemic circulation (for liver metastasis), and only a manifold reduced dose of the crosslinking agent is delivered to the liver.

When injected intratumorally at the original dose (1 mg per therapy point), DNAmix has an impact only on a small portion of the affected tissue. In this case, only some cancer stem cells are exposed to the eradication effect of medications.

The proliferation of dormant metastases can be activated when the therapeutic effect of the drugs is incomplete (i.e., the cytostatic agent has induced crosslinks, but the DNAmix failed to reach this nidus at the proper time). The interstrand crosslinks in cancer stem cells will be repaired, the cells will not acquire apoptotic status, and so-called clonal selection will occur (58,59). This situation will result in additional tumor progression.

Therefore, we inferred that it is reasonable to increase the drug dose (to a dose close to the one used in mouse experiments) and optimize the regimen of DNAmix delivery. The dose of DNAmix was increased to 12 mg in 12 mL of saline. Additionally, a decision was made that instead of trying systemic administration of DNAmix, injections should be made into lymph depots within inguinal and axillary lymph node clusters, as well as at a distance of 5 to 7 cm to the right of the umbilicus (paraumbilically); injections in the most prominent local tumor nidi are prioritized. It is anticipated that DNAmix is injected into lymph depot areas, therapeutic DNA fragments will spread along lymph flow in an ascending direction, embracing the target areas of metastatic tumor nidi. When administered into the bloodstream, DNA is immediately degraded and loses its therapeutic effect (60).

The adjusted regimen was used, starting September 2023, for patient 12 with carcinomatous pleurisy, and an immediate favorable clinical response was achieved. The standard regimen was used for this patient during the first three courses: 1 mg of DNAmix was administered locally (intratumorally); injections were made into the most prominent tumor areas. Starting with the fourth therapy course, we used the new therapeutic regimen when high-dose DNAmix was injected into the lymph depot areas and the pleural cavity. Before switching to a different therapy regimen, the patient suffered the most from dyspnea caused by the accumulation of fluid in the pleural cavities and the need to drain the fluid by puncture. After starting the new drug regimen, the severity of the patient’s dyspnea decreased, making frequent invasive procedures unnecessary and having a beneficial impact on her quality of life.

We demonstrated that the Karanahan technology also activates the adaptive antitumor immune response, which undoubtedly favorably affects treatment.

The key technical issues resolved during the transfer of the laboratory protocol of the technology into clinical practice

The key technical issues associated with the technology transfer have been resolved during project implementation.

• A patient enrollment protocol has been developed. The accrual rate of patients meeting the inclusion criteria indicates that future studies will necessitate the recruitment of large cancer treatment centers of federal level.
• The technical procedures employed in the technology require appropriate specialized instrumentation and a team of professional cellular and molecular biologists.
• The technique for collecting and transporting resected material have been successfully mastered.
• A stepwise protocol for assessing the technology parameters has been elaborated, including procedures for determining the duration of the repair cycle and the final point of treatment.
• The following parameters have been optimized: the dose was increased and the topology of DNAmix administration was expanded. The paragraph “monitoring the development of systemic inflammatory response” has been added to the protocol. Specific blood chemistry parameters being indicative of incipient systemic inflammatory response and necessitating chronometric monitoring have been identified. A detoxification regimen to manage the onset of systemic inflammatory response symptoms has been developed.

All the issues solved will be incorporated into the final protocol of the randomized clinical trial.

Uncertainties and limitations of the technology

This study had several uncertainties and limitations; some of them have been solved throughout the trial:

• The lack of prior studies on the topic: no data on the optimal design of the experimental and clinical work were available as it was a pioneering study.
• Sample size.
• Technical challenges in achieving the delivery of the DNAmix preparation to all the cancer stem cells. Upon 100% delivery, all cancer stem cells are eradicated and full cure is achieved as it observed in the murine models (31,33,34,36). This challenge is partially resolved by making multiple low-volume injections of the required DNAmix doses into accessible tumor growth sites, as well as injection of DNAmix preparation into the peritoneal cavity, the pleural cavity, and metastatic foci.
• The infeasibility of determining the biological clock of small metastases, which may differ from the primary tumor or a larger metastatic focus, which can potentially reduce therapeutic efficacy.

Conclusions

The conducted clinical trials provide evidence for the feasibility and practical applicability of transferring the Karanahan technology to clinical practice.

Initial data on the potential clinical significance of the novel approach have been obtained; however, these findings need to be validated in large-scale randomized clinical trials.

An unexpected observation was conceptualized: the technology applied as described in this study effectively “shifts the temporal reference point of disease progression to an earlier stage”. On the basis of these findings, it is fair to say that comprehensive application of the technology, when the DNA preparation reaches all tumor nidi, changes the tumor status and makes it operable. Furthermore, in the case of positive dynamics, it is possible to return to medications that had to be ceased because of ineffectiveness due to generalization and terminal progression of the neoplasm.

The conducted studies have identified several parameters that will be mandatorily taken into account in a comprehensive large-scale trial.

• The dose of the DNAmix preparation needs to be escalated from 1 to ≥12 mg.
• The DNAmix preparation should be administered not only intratumorally but also be injected into the lymph depots (inguinal lymph node clusters, axillary lymph nodes, and paraumbilically).
• Monitoring of C-reactive protein levels during Karanahan therapy is essential. Upon detection of signs indicating a systemic inflammatory response, detoxification measures must be initiated to prevent its progression.

Acknowledgments

The authors express their gratitude to the Common Use Center of Flow Cytometry IC&G SB RAS and the Common Use Center for Microscopy of Biologic Objects SB RAS.

Footnote

Reporting Checklist: The authors have completed the TREND reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2722/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2722/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2722/prf

Funding: This study was supported by the Russian Ministry of Science and High Education via the Institute of Cytology and Genetics (No. FWNR-2026-0025), and Inga N. Zaitseva.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2722/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Local Ethics Committee of the Research Institute of Fundamental and Clinical Immunology (protocol No. 120 of November 07, 2019). Written informed consent to participate in the study was obtained from each of the patients, which specified open publication of the results presented as reports or otherwise.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Riggio AI, Varley KE, Welm AL. The lingering mysteries of metastatic recurrence in breast cancer. Br J Cancer 2021;124:13-26. [Crossref] [PubMed]
2. Schepotin IB, Zotov AS, Lyubota RV, et al. The clinical significance of breast cancer stem cells (review of literature). Tumors Female Reprod Syst 2014;14-9.
3. Schatton T, Murphy GF, Frank NY, et al. Identification of cells initiating human melanomas. Nature 2008;451:345-9. [Crossref] [PubMed]
4. Shurin GV, Tourkova IL, Kaneno R, et al. Chemotherapeutic agents in noncytotoxic concentrations increase antigen presentation by dendritic cells via an IL-12-dependent mechanism. J Immunol 2009;183:137-44. [Crossref] [PubMed]
5. Schofield P, Carey M, Love A, et al. ‘Would you like to talk about your future treatment options?’ Discussing the transition from curative cancer treatment to palliative care. Palliat Med 2006;20:397-406. [Crossref] [PubMed]
6. Kim WT, Ryu CJ. Cancer stem cell surface markers on normal stem cells. BMB Rep 2017;50:285-98. [Crossref] [PubMed]
7. Schito L, Rey S, Xu P, et al. Metronomic chemotherapy offsets HIFα induction upon maximum‐tolerated dose in metastatic cancers. EMBO Mol Med 2020;12:e11416. [Crossref] [PubMed]
8. Sell S. Alpha-fetoprotein, stem cells and cancer: how study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer. Tumour Biol 2008;29:161-80. [Crossref] [PubMed]
9. Takeda K, Mizushima T, Yokoyama Y, et al. Sox2 is associated with cancer stem-like properties in colorectal cancer. Sci Rep 2018;8:17639. [Crossref] [PubMed]
10. Wicha MS. Cancer stem cell heterogeneity in hereditary breast cancer. Breast Cancer Res 2008;10:105. [Crossref] [PubMed]
11. Tashireva LA, Perelmuter VM, Manskikh VN, et al. Types of Immune-Inflammatory Responses as a Reflection of Cell-Cell Interactions under Conditions of Tissue Regeneration and Tumor Growth. Biochemistry (Mosc) 2017;82:542-55. [Crossref] [PubMed]
12. Van Cutsem E, Nordlinger B, Cervantes A, et al. Advanced colorectal cancer: ESMO Clinical Practice Guidelines for treatment. Ann Oncol 2010;21:v93-7. [Crossref] [PubMed]
13. Wang J, Liu X, Jiang Z, et al. A novel method to limit breast cancer stem cells in states of quiescence, proliferation or differentiation: Use of gel stress in combination with stem cell growth factors. Oncol Lett 2016;12:1355-60. [Crossref] [PubMed]
14. Wright MH, Calcagno AM, Salcido CD, et al. Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res 2008;10:R10. [Crossref] [PubMed]
15. Su J, Wu S, Wu H, et al. CD44 is functionally crucial for driving lung cancer stem cells metastasis through Wnt/β-catenin-FoxM1-Twist signaling. Mol Carcinog 2016;55:1962-73. [Crossref] [PubMed]
16. Wu C, Wei Q, Utomo V, et al. Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. Cancer Res 2007;67:8216-22. [Crossref] [PubMed]
17. Wu J, Waxman DJ. Metronomic cyclophosphamide eradicates large implanted GL261 gliomas by activating antitumor Cd8(+) T-cell responses and immune memory. Oncoimmunology 2015;4:e1005521. [Crossref] [PubMed]
18. Yilmaz OH, Morrison SJ. The PI-3kinase pathway in hematopoietic stem cells and leukemia-initiating cells: a mechanistic difference between normal and cancer stem cells. Blood Cells Mol Dis 2008;41:73-6. [Crossref] [PubMed]
19. Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153-66. [Crossref] [PubMed]
20. Murakawa Y, Sakayori M, Otsuka K. Impact of palliative chemotherapy and best supportive care on overall survival and length of hospitalization in patients with incurable Cancer: a 4-year single institution experience in Japan. BMC Palliat Care 2019;18:45. [Crossref] [PubMed]
21. Yin T, Wei H, Gou S, et al. Cancer stem-like cells enriched in Panc-1 spheres possess increased migration ability and resistance to gemcitabine. Int J Mol Sci 2011;12:1595-604. [Crossref] [PubMed]
22. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009;457:608-11. [Crossref] [PubMed]
23. Yin W, Wang J, Jiang L, et al. Cancer and stem cells. Exp Biol Med (Maywood) 2021;246:1791-801. [Crossref] [PubMed]
24. Yu F, Yao H, Zhu P, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007;131:1109-23. [Crossref] [PubMed]
25. Garcia-Mayea Y, Mir C, Masson F, et al. Insights into new mechanisms and models of cancer stem cell multidrug resistance. Semin Cancer Biol 2020;60:166-80. [Crossref] [PubMed]
26. Yuan F, Shi H, Ji J, et al. Capecitabine metronomic chemotherapy inhibits the proliferation of gastric cancer cells through anti-angiogenesis. Oncol Rep 2015;33:1753-62. [Crossref] [PubMed]
27. Zhang Z, Zhang X, Newman K, et al. MicroRNA regulation of oncolytic adenovirus 6 for selective treatment of castration-resistant prostate cancer. Mol Cancer Ther 2012;11:2410-8. [Crossref] [PubMed]
28. Badalyan Gk, Badalyan L. Evaluation of the effectiveness of palliative chemotherapy depending on the intensity of treatment regimens. Eurasian J Oncol 2012;:413.
29. Eramo A, Lotti F, Sette G, et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 2008;15:504-14. [Crossref] [PubMed]
30. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 2009;347:70-8. [Crossref] [PubMed]
31. Ruzanova V, Proskurina A, Efremov Y, et al. Chronometric Administration of Cyclophosphamide and a Double-Stranded DNA-Mix at Interstrand Crosslinks Repair Timing, Called “Karanahan” Therapy, Is Highly Efficient in a Weakly Immunogenic Lewis Carcinoma Model. Pathol Oncol Res 2022;28:1610180. [Crossref] [PubMed]
32. Proskurina AS, Kupina VV, Efremov YR, et al. Karanahan: A Potential New Treatment Option for Human Breast Cancer and Its Validation in a Clinical Setting. Breast Cancer (Auckl) 2022;16:11782234211059931. [Crossref] [PubMed]
33. Potter EA, Dolgova EV, Proskurina AS, et al. A strategy to eradicate well-developed Krebs-2 ascites in mice. Oncotarget 2016;7:11580-94. [Crossref] [PubMed]
34. Potter EA, Proskurina AS, Ritter GS, et al. Efficacy of a new cancer treatment strategy based on eradication of tumor-initiating stem cells in a mouse model of Krebs-2 solid adenocarcinoma. Oncotarget 2018;9:28486-99. [Crossref] [PubMed]
35. Potter EA, Ritter GS, Dolgova EV, et al. Evaluating the efficiency of the tumor-initiating stem cells eradication strategy on the example of ascite form of mouse gepatocarcinoma G-29. Vopr Onkol 2018;64:818-29.
36. Kisaretova PE, Kirikovich SS, Ritter GS, et al. Approbation of the Cancer Treatment Approach Based on the Eradication of TAMRA+ Cancer Stem Cells in a Model of Murine Cyclophosphamide Resistant Lymphosarcoma. Anticancer Res 2020;40:795-805. [Crossref] [PubMed]
37. Ruzanova VS, Proskurina AS, Ritter GS, et al. Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies. Anticancer Res 2021;41:3371-87. [Crossref] [PubMed]
38. Ruzanova V, Proskurina A, Ritter G, et al. The synergistic antitumor effect of Karanahan technology and in situ vaccination using anti-OX40 antibodies. Oncol Res 2025;33:1229-48. [Crossref] [PubMed]
39. Dolgova EV, Potter EA, Proskurina AS, et al. Evaluating the effectiveness of the tumor-initiating stem cells eradication strategy on the example of human glioblastoma cell line U87. Vopr Onkol 2019;65:904-19.
40. Dolgova EV, Andrushkevich OM, Kisaretova PE, et al. Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma. Cancer Biol Med 2021;18:910-30. [Crossref] [PubMed]
41. Proskurina AS, Ruzanova VS, Ostanin AA, et al. Theoretical premises of a “three in one” therapeutic approach to treat immunogenic and nonimmunogenic cancers: a narrative review. Transl Cancer Res 2021;10:4958.
42. Desai AP, Go RS, Poonacha TK. Category of evidence and consensus underlying National Comprehensive Cancer Network guidelines: Is there evidence of progress? Int J Cancer 2021;148:429-36. [Crossref] [PubMed]
43. Zagouri F, Liakou P, Bartsch R, et al. Discrepancies between ESMO and NCCN breast cancer guidelines: An appraisal. Breast 2015;24:513-23. [Crossref] [PubMed]
44. Poonacha TK, Go RS. Level of scientific evidence underlying recommendations arising from the National Comprehensive Cancer Network clinical practice guidelines. J Clin Oncol 2011;29:186-91. [Crossref] [PubMed]
45. Gradishar WJ, Moran MS, Abraham J, et al. NCCN Guidelines® Insights: Breast Cancer, Version 5.2025. J Natl Compr Canc Netw 2025;23:426-36. [Crossref] [PubMed]
46. Gennari A, André F, Barrios CH, et al. ESMO Clinical Practice Guideline for the diagnosis, staging and treatment of patients with metastatic breast cancer. Ann Oncol 2021;32:1475-95. [Crossref] [PubMed]
47. Cherny NI, Oosting SF, Dafni U, et al. ESMO-Magnitude of Clinical Benefit Scale version 2.0 (ESMO-MCBS v2.0). Ann Oncol 2025;36:866-908. [Crossref] [PubMed]
48. Dolgova EV, Alyamkina EA, Efremov YR, et al. Identification of cancer stem cells and a strategy for their elimination. Cancer Biol Ther 2014;15:1378-94. [Crossref] [PubMed]
49. Pearson N, Naylor PJ, Ashe MC, et al. Guidance for conducting feasibility and pilot studies for implementation trials. Pilot Feasibility Stud 2020;6:167. [Crossref] [PubMed]
50. Rajadhyaksha V. Conducting feasibilities in clinical trials: an investment to ensure a good study. Perspect Clin Res 2010;1:106-9.
51. Bogachev S, Dolgova E, Potter E, et al. A method for the treatment of cancer. Russia; RU 2662354 C1. 2018.
52. Likhacheva AS, Rogachev VA, Nikolin VP, et al. Involvement of exogenous DNA in the molecular processes in somatic cell. Informatsionnyy Vestn VOGiS Her Vavilov Soc Genet Breed 2008;12:426-73.
53. Berthault N, Bergam P, Pereira F, et al. Inhibition of DNA Repair by Inappropriate Activation of ATM, PARP, and DNA-PK with the Drug Agonist AsiDNA. Cells 2022;11:2149. [Crossref] [PubMed]
54. Tronov VA, Kramarenko II, Karpukhin AV. Colon cancer: repair deficiency, genome instability, apoptosis resistance, disease risk assessment. Vopr Onkol 2005;51:159-66.
55. Dolgova EV, Proskurina AS, Nikolin VP, et al. ‘Delayed death’ phenomenon: A synergistic action of cyclophosphamide and exogenous DNA. Gene 2012;495:134-45. [Crossref] [PubMed]
56. Dolgova EV, Efremov YR, Orishchenko KE, et al. Delivery and processing of exogenous double-stranded DNA in mouse CD34+ hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA. Gene 2013;528:74-83. [Crossref] [PubMed]
57. Alyamkina EA, Nikolin VP, Popova NA, et al. Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts. Cancer Cell Int 2015;15:32. [Crossref] [PubMed]
58. Salavaty A, Azadian E, Naik SH, et al. Clonal selection parallels between normal and cancer tissues. Trends Genet 2023;39:358-80. [Crossref] [PubMed]
59. Greaves M, Maley CC. Clonal evolution in cancer. Nature 2012;481:306-13. [Crossref] [PubMed]
60. Dolgova EV, Rogachev VA, Nikolin VP, et al. Leukocyte stimulation by DNA fragments shored up protamine in cyclophosphamide-induced leukopoiesis in mice. Vopr Onkol 2009;55:761-4.