An Overview of Introductory and Advanced Survival Analysis Methods in Clinical Applications: Where Have we Come so far?

The time-to-event relationship for survival modeling is considered when designing a study in clinical trials. However, because time-to-event data are mostly not normally distributed, survival analysis uses non-parametric data processing and analysis methods, mainly Kaplan-Meier (KM) estimation models and Cox proportional hazards (CPH) regression models. At the same time, the log-rank test can be applied to compare curves from different groups. However, resorting to conventional survival analysis when fundamental assumptions, such as the Cox PH assumption, are not met can seriously affect the results, rendering them flawed. Consequently, it is necessary to examine and report more sophisticated statistical methods related to the processing of survival data, but at the same time, able to adequately respond to the contemporary real problems of clinical applications. On the other hand, the frequent misinterpretation of survival analysis methodology, combined with the fact that it is a complex statistical tool for clinicians, necessitates a better understanding of the basic principles underlying this analysis to effectively interpret medical studies in making treatment decisions. In this review, we first consider the basic models and mechanisms behind survival analysis. Then, due to common errors arising from the inappropriate application of conventional models, we revise more demanding statistical extensions of survival models related to data manipulation to avoid wrong results. By providing a structured review of the most representative statistical methods and tests covering contemporary survival analysis, we hope this review will assist in solving problems that arise in clinical applications.

Personalized dendritic cell vaccination in cancer therapy: An evidence-based research study.

Background: Although chemotherapy is considered to be the golden standard, it does not come without a price. Toxicities and resistance are frequently limiting its effectiveness. Immunotherapy has emerged as a safer therapeutic alternative but still has a long way until it has proven to be of equal efficacy. A type of immunotherapy is dendritic cell (DC) vaccination. Aims and Objectives: We have developed a novel platform for the generation of autologous DCs that have been activated against peptides that are personalized for each patient individually. The aim of the study was to clinically evaluate this platform. Materials and Methods: Our platform and our algorithm for the determination of the immunogenic peptides has been tested. DC generation was verified both morphologically and by CD80/86 expression. Peptide antigenicity was determined using a number of T-cell epitope prediction algorithms. Response to therapy was evaluated using response evaluation criteria in solid tumors (RECIST) criteria by the doctors involved. Immune status was also evaluated before and after DC vaccination and correlated with circulated tumor cell count. Results: It was found that DC vaccine increased immune activation while correlated with decreased circulating tumor cell counts. Clinical evaluation by the determination of immune markers may be a superior tool than using RECIST criteria. Conclusion: Dendritic cell therapies could prove to be a valuable tool in cancer treatment.

Is Network Meta-analysis a Revolutionary Statistical Tool for Improving the Reliability of Clinical Trial Results? A Brief Overview and Emerging Issues Arising.

Network meta-analysis (NMA) as the quantification of pairwise meta-analysis in a network format has been of particular interest to medical researchers in recent years. As a powerful tool with which direct and indirect evidence from multiple interventions can be synthesized simultaneously in the study and design of clinical trials, NMA enables inferences to be drawn about the relative effect of drugs that have never been compared. In this way, NMA provides information on the hierarchy of competing interventions for a given disease concerning clinical effectiveness, thus giving clinicians a comprehensive picture for decision-making and potential avoidance of additional costs. However, estimates of treatment effects derived from the results of network meta-analyses should be interpreted with due consideration of their uncertainty, because simple scores or treatment probabilities may be misleading. This is particularly true where, given the complexity of the evidence, there is a serious risk of misinterpretation of information from aggregated data sets. For these reasons, NMA should be performed and interpreted by both expert clinicians and experienced statisticians, while a more comprehensive search of the literature and a more careful evaluation of the body of evidence can maximize the transparency of the NMA and potentially avoid errors in its interpretation. This review provides the key concepts as well as the challenges we face when studying a network meta-analysis of clinical trials.

CTLs, NK cells and NK-derived EVs against breast cancer.

Patients with advanced stage breast cancer need novel therapies. New potential treatments have been developed, such as adoptive cellular therapies and alternative cell-free immunotherapies. The goal of this study was to assess the cytotoxicity of three of the patient-derived immune components, CTLs, NK cells and NK-derived EVs, and evaluate the potential for the development of novel therapy against breast cancer. CTLs were activated against MUC-1 antigen. The in vitro cytotoxic activity of three components was assessed with flow cytometry and in vivo study revealed the efficacy of adoptive cell therapy. Overall, CTLs exhibited the highest cytotoxicity against spheroids of MCF7 breast adenocarcinoma, reaching in all cases higher than double the percentage of NK cells' cytotoxicity. NK-derived EVs exhibited the lowest effect against MCF7 spheroids comparing to the two cell populations. MUC-1 specific CTLs were evaluated with adoptive cell therapy mice study and appeared to be well tolerable and moderately efficacious. More studies need to be performed with CTLs to evaluate safety and efficacy in order to assess their clinical potential, while NK cells and NK-derived EVs are promising candidates that require more experiments to enhance their cytotoxicity.

Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo.

Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents (adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside). Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS), tumor suppressors (TP53, WT1), and others (GSTP1, HSP90, proliferation rate), in 59 tumor cell lines of the National Cancer Institute (NCI, USA), indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin's potential for drug combination regimens.

Supportive Oligonucleotide Therapy (SOT) as a Potential Treatment for Viral Infections and Lyme Disease: Preliminary Results.

Antisense therapy is widely used as an alternative therapeutic option for various diseases. RNA interference might be effective in infections, through the degradation of messenger RNA and, therefore, translation process. Hence, proteins essential for microorganisms and viruses' proliferation and metabolism are inhibited, leading to their elimination. The present study aimed to evaluate the use of oligonucleotide in patients infected by Epstein-Barr (EBV) or Herpes Simplex Viruses 1/2 or with Lyme Disease caused by Borrelia burgdorferi. Blood samples were collected from 115 patients and the different species were characterized using molecular biology techniques. Then, SOT molecules (Supportive Oligonucleotide Therapy), which are specific small interfering RNA (siRNA), were designed, produced, and evaluated, for each specific strain. Oligonucleotides were administered intravenously to patients and then a quantitative Polymerase Chain Reaction was used to evaluate the effectiveness of SOT. This study revealed that for Lyme Disease, one or two SOT administrations can lead to a statistically significant decrease in DNA copies, while for viruses, two or three administrations are required to achieve a statistically significant reduction in the genetic material. These preliminary results indicate that antisense SOT therapy can be considered a potential treatment for viral as well as Lyme diseases.

Adoptive transfer of activated immune cells against solid tumors: A preliminary study.

BACKGROUND: This study presents preliminary results concerning the effectiveness of a novel immunotherapy in cancer. The proposed adoptive cellular therapy product contains a mixture of effector immune cells, specifically macrophages, NK cells, dendritic cells, cytotoxic T lymphocytes and monoclonal antibody producing plasma cells. METHODS: The results were based on both descriptive and inferential statistical analysis of data concerning 17 cancer patients. Particularly, performance scales such as clinical condition, Karnofsky-Index, ECOG index and symptom's scale were evaluated post therapy administration (4 months). Furthermore, circulating tumor cells (CTCs) and a specific tumor marker (EpCAM) were measured pre- and post-cellular therapy. RESULTS: The results revealed a positive evaluation for clinical condition (70.59 %), Karnofsky-Index (88.23 %), ECOG index (94.12 %), and symptoms' scale (64.70 %). In addition, statistically significant reductions were found for both CTCs (p = 0.0016) and EpCAM positive cells (p = 0.0005), post-therapy, which were related to large size effects, namely 0.77 and 0.85, respectively. No cytokine storm, anaphylaxis or severe adverse events were observed with 4 months follow up evaluation. CONCLUSIONS: These preliminary results indicate that the proposed cellular therapy can be considered for further studies in clinical trials.

A novel platform for the production of autologous human antibodies.

At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient's own cells, bringing personalized, targeted therapy for cancer one step closer.

Teaching an old dog new tricks: The case of Fenbendazole.

The objective of this study is the assessment of the cytotoxic effect of fenbendazole and its commercially available formulation, which is used for its antihelmintic properties. The formulation was tested for its efficacy as well as the determination of the ingredients with proliferation assays and analytical techniques. HPLC, LC-MS and NMR confirmed the stated amount of active ingredient on the label. Dissolution studies were performed to simulate the ability of fenbendazole to dissolve adequately in the fluids of the Gastrointestinal tract, be absorbed in the circulation and reach certain areas of the human body. However, dissolution studies showed that both brands possess issues in their distribution. The in vitro drug screening exhibited potential cytotoxic effect in different types of human cancer cell lines and MDA-MB-231 human breast adenocarcinoma cells appeared to be the most sensitive with IC50 value lower than 10 µM.

Adoptive Cellular Transfer Immunotherapies for Cancer.

Patients with cancer require efficient treatment approaches as the mortality rate due to their disease is high. Conventional therapies, like chemotherapy and radiation, have severe side effects. Drug discovery is focusing on the development of alternative strategies that could have beneficial effects to the patients. Cellular therapies are potential therapeutics, and the generation of new products is growing fast. The concept involves the isolation of immune cells, ex vivo activation and reinfusion into the patient. The goal is to boost the immune cells to fight cancer cells. Different immune cells can be used, including dendritic cells, T cells, NK cells, macrophages and B lymphocytes. Some products have already gained FDA approval, while many more are currently in clinical trials. Research is focusing on the improvement of the function of the cells that may require genetic modification or combination with other therapies. Finally, it is crucial to develop novel technologies that could be used in monitoring of the immune profile of patients that have received a cellular therapy to assess the efficacy of the treatment.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases.

BACKGROUND: Patients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs). METHODS: In this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected. RESULTS: Our analyses resulted in evaluations of 94.12% (95% CI 0.71-0.99) for sensitivity, 5.26% (95% CI 0.01-0.26) for specificity, a predictive value of 47.06% (95% CI 0.29-0.65) for a positive response, a predictive value of 50% (95% CI 0.01-0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30-0.64). CONCLUSIONS: This is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

An Electrostatically-steered Conformational Selection Mechanism Promotes SARS-CoV-2 Spike Protein Variation.

After two years since the outbreak, the COVID-19 pandemic remains a global public health emergency. SARS-CoV-2 variants with substitutions on the spike (S) protein emerge increasing the risk of immune evasion and cross-species transmission. Here, we analyzed the evolution of the S protein as recorded in 276,712 samples collected before the start of vaccination efforts. Our analysis shows that most variants destabilize the S protein trimer, increase its conformational heterogeneity and improve the odds of the recognition by the host cell receptor. Most frequent substitutions promote overall hydrophobicity by replacing charged amino acids, reducing stabilizing local interactions in the unbound S protein trimer. Moreover, our results identify "forbidden" regions that rarely show any sequence variation, and which are related to conformational changes occurring upon fusion. These results are significant for understanding the structure and function of SARS-CoV-2 related proteins which is a critical step in vaccine development and epidemiological surveillance.

Supportive Oligonucleotide Therapy (SOT) as an Alternative Treatment Option in Cancer: A Preliminary Study.

BACKGROUND/AIM: An early evaluation concerning the effectiveness of supportive oligonucleotide therapy (SOT) in cancer as a monotherapy and in combination with other types of treatment. PATIENTS AND METHODS: This study evaluated the clinical condition and performance status (Karnofsky-Index) of 95 patients, post-SOT administration. Furthermore, circulating tumor cells (CTCs) from 47 patients' pre- and post-SOT administration were measured and analyzed by repeated-measures ANOVA. RESULTS: Improvement of the clinical condition was observed in all patients who used SOT (77.89%), SOT in combination with other therapy (69.77%) and SOT as a monotherapy or no information was given concerning another therapy (84.31%). Positive results for Karnofsky-Index were also observed in 71.58%, 61.36%, and 80.39%, respectively. Finally, statistically significant reductions in CTCs were observed for both SOT as a monotherapy and SOT as an adjunctive therapy. CONCLUSION: The preliminary results indicate that SOT therapy can be used both as monotherapy as well as in combination with other therapies for cancer.

A Novel Method for Colorectal Cancer Screening Based on Circulating Tumor Cells and Machine Learning.

Colorectal cancer is one of the most common types of cancer, and it can have a high mortality rate if left untreated or undiagnosed. The fact that CRC becomes symptomatic at advanced stages highlights the importance of early screening. The reference screening method for CRC is colonoscopy, an invasive, time-consuming procedure that requires sedation or anesthesia and is recommended from a certain age and above. The aim of this study was to build a machine learning classifier that can distinguish cancer from non-cancer samples. For this, circulating tumor cells were enumerated using flow cytometry. Their numbers were used as a training set for building an optimized SVM classifier that was subsequently used on a blind set. The SVM classifier's accuracy on the blind samples was found to be 90.0%, sensitivity was 80.0%, specificity was 100.0%, precision was 100.0% and AUC was 0.98. Finally, in order to test the generalizability of our method, we also compared the performances of different classifiers developed by various machine learning models, using over-sampling datasets generated by the SMOTE algorithm. The results showed that SVM achieved the best performances according to the validation accuracy metric. Overall, our results demonstrate that CTCs enumerated by flow cytometry can provide significant information, which can be used in machine learning algorithms to successfully discriminate between healthy and colorectal cancer patients. The clinical significance of this method could be the development of a simple, fast, non-invasive cancer screening tool based on blood CTC enumeration by flow cytometry and machine learning algorithms.

Targeting SARS-CoV-2 Polymerase with New Nucleoside Analogues.

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.

Genistein as a dietary supplement; formulation, analysis and pharmacokinetics study.

The objective of this study is to improve and optimize the formulation of Genistein in capsules in order to result in a better pharmacokinetic profile comparing to existing commercial products. In order to do this, five different formulations of Genistein capsules were developed and examined by reviewing their disintegration and dissolution properties. Furthermore, flowability of the powder along with potent incompatibilities between Genistein and its excipients were monitored through their thermal properties. The final formulation of Genistein was quantified using HPLC analysis and then its stability was evaluated thoroughly in real time and accelerated conditions. Finally, with the target to have a product with actual results, in vitro and in vivo studies were conducted. The final product proved to have better results in disintegration and dissolution. Moreover, R.G.C.C.'s capsules exhibited enhanced action in human cell lines as well as impressive pharmacokinetic results in animal models. The in vitro results showed an advantage of the R.G.C.C. product compared to the commercial one, whereas its maximum concertation in vivo was determined 34% higher than the commercial one.

Cancer Comprehensive Analysis in Gastric Carcinoma: Benefits and New Perspectives.

Gastric cancer is one of the most common and deadly cancers worldwide. Screening tests as well as tools for prediction of treatment outcomes and prognosis have been developed, but they have many limitations. The integration of liquid biopsy provided new aspects in screening and diagnosis of gastric cancer. In the present study, we used different techniques, studying the genetic and epigenetic profile of circulating tumor cells. We aimed to acquire all the available information, compare it with already existing studies, and evaluate the benefit of this approach. A blood sample was isolated from 2 gastric cancer patients at stages III-IV, followed by the isolation of CTCs. The circulating tumor cells were used for array comparative genomic hybridization, next-generation sequencing, and whole gene expression microarrays. Different variants were detected, while the microsatellite instability status was stable in both cases. The tumor mutational burden was low to medium. Gene expression assays revealed that >100 genes were overexpressed compared to noncancer samples. Amplifications of X chromosome were also observed in both cases, by using array comparative genomic hybridization. Although there are several techniques for cancer screening, prediction of therapy outcomes, and prognosis, the application of a complete comprehensive cancer panel, combining the study of variants, fusions, chromosomal abnormalities, and gene expression, is more appropriate. Information provided by the above techniques might contribute in designing more efficient treatment protocols and screening tools. Despite the limitation of samples, the data are encouraging, and further study is needed so that they can be used at clinical level.

Increased breast cancer cell sensitivity to cisplatin using a novel small molecule inhibitor.

BACKGROUND: Although cisplatin is used for the treatment of more than half of cancer patients, its use is restricted by serious side effects as well as the development of cisplatin-resistant cancer cells, limiting its use. In RGCC we have synthesized an intermediate molecule in an ERK inhibitor synthesis process. AIMS AND OBJECTIVES: The aim of the study was to evaluate the effects of combined cisplatin plus RGCC molecule treatment on MCF-7 and MDAMB231 breast cancer cell viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression. MATERIALS AND METHODS: Cell viability and proliferation were measured by Crystal violet exclusion dye and MTT respectively. Clone formation and wound healing assays were also used for clone formation and cell migration evaluation. Cell cycle was studied by flow cytometry, expression of genes was evaluated by PCR and protein expression was evaluated by western blot. RESULTS: It was found that combination therapy decreased cell viability and proliferation, caused growth arrest, decreased cancer cell invasiveness and the ability to form clones as well as perturbed the expression of genes involved in ERK, cell cycle and cell death pathways. Conclusion: Although the exact mechanism of action of the combination therapy remains to be investigated, it was found that it is more effective than cisplatin monotherapy. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.

Evaluation of Tegaran Formula ZhenHua cytotoxicity against human cancer cell lines.

The aim of this study is to evaluate the potential health effects of Tegaran Formula ZhenHua, a nutritional supplement used mainly by cancer patients. Its active ingredients and cytotoxicity was assessed with analytical methods and viability assays, respectively. The analytical methods consisted of dissolution, disintegration, HPLC, LC/MS, GC/MS and NMR. Cytotoxicity was assessed by MTT, SRB, CVE colorimetric viability assays in 0, 24, 48 and 72h time points. The results indicate that Tegaran Formula ZhenHua supplement did not present any cytotoxic effects due to issues related to the capsules' solubility, distribution and identification of the active ingredient.

Structural models of human ACE2 variants with SARS-CoV-2 Spike protein for structure-based drug design.

Emergence of coronaviruses poses a threat to global health and economy. The current outbreak of SARS-CoV-2 has infected more than 28,000,000 people and killed more than 915,000. To date, there is no treatment for coronavirus infections, making the development of therapies to prevent future epidemics of paramount importance. To this end, we collected information regarding naturally-occurring variants of the Angiotensin-converting enzyme 2 (ACE2), an epithelial receptor that both SARS-CoV and SARS-CoV-2 use to enter the host cells. We built 242 structural models of variants of human ACE2 bound to the receptor binding domain (RBD) of the SARS-CoV-2 surface spike glycoprotein (S protein) and refined their interfaces with HADDOCK. Our dataset includes 140 variants of human ACE2 representing missense mutations found in genome-wide studies, 39 mutants with reported effects on the recognition of the RBD, and 63 predictions after computational alanine scanning mutagenesis of ACE2-RBD interface residues. This dataset will help accelerate the design of therapeutics against SARS-CoV-2, as well as contribute to prevention of possible future coronaviruses outbreaks.