To estimate oxidative stress and DNA damage in postCOVID patients

Title: To estimate oxidative stress and DNA damage in Post COVID patients. Background There are a subset of COVID-19 patients who develop sequelae to the disease and oxidative stress is a less studied factor in the development of the sequelae. Aims and Objectives: We have estimated levels of lipid peroxidation (LPO) via malondialdehyde assay kit and DNA damage via alkaline comet assay in hospitalized post-COVID patients symptomatic 4 weeks after testing RT-PCR positive and studied their clinical radiological correlation as a means of estimating the oxidative stress in them. Method(s): It was a single-center, hospital-based comparative case-control pilot study in which 40 post-COVID-19 patients and 40 healthy controls were enrolled. The residual symptoms and baseline clinical and radiological profile of the subjects were also assessed and lipid peroxidation and DNA comet analysis were performed in the blood samples of patients and controls. Result(s): Mean value of LPO was increased (1155.9 +/- 204.82 nmol/ml) in post COVID subjects as compared to controls (715.5 +/- 85.51nmole/ml (P=0.0405). Values were directly proportional to the Severity of COVID (P=0.0317) and X-ray severity score(P=0.009) and were found higher in patients with comorbidities (P=0.0320) and multisystem involvement specifically in those developing a neurological sequela (P=0.0083). Damaged DNA tails and the tailing is directly proportional to DNA damage. The comet parameters measured in our study were Tail length, Tail DNA (%), and Olive tail moment. All these comet parameters were found elevated in Post COVID subjects as compared with healthy controls. Conclusion(s): Oxidative stress and DNA damage, has a role in the development of post-COVID sequelae as seen by high levels of LPO and tail DNA in these subjects.

Clinical Investigation of Leukocyte DNA Damage in COVID-19 Patients.

This prospective cross-sectional study aimed to evaluate leukocyte DNA damage in coronavirus disease (COVID-19) patients. In this study, 50 COVID-19-positive patients attending the Erzurum City Hospital Internal Medicine Outpatient Clinic and 42 control group patients were included. DNA damage was detected in living cells through leukocyte isolation in 50 COVID-19-positive patients using the comet assay method. DNA tail/head (olive) moments were evaluated and compared. White blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), neutrophils (NEU), lymphocytes (LYM), eosinophils (EO), monocytes (MONO), basophils (BASO), platelets (PLT), and the neutrophil/lymphocyte ratio (NLR) were analyzed. The RBC, lymphocyte, eosinophil, and monocyte means were significantly higher in the control group (p < 0.05), whereas the HGB and neutrophile means were significantly higher in the study group (p < 0.05). There were significant negative correlations between COVID-19 and RBC (r = -0.863), LYM (r = -0.542), EO (r = -0.686), and MONO (r = -0.385). Meanwhile, there were significant positive correlations between COVID-19 and HGB (r = 0.863), NEU (r = 0.307), tail moment (r = 0.598), and olive moment (r = 0.582). Both the tail and olive moment mean differences were significantly higher in the study group, with higher ranges (p < 0.05). COVID-19 infection caused statistically significant increases in both the tail and olive damage percentage in patients, causing DNA damage. Lastly, the NLR rate was associated with the presence and progression of COVID-19.

Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells.

Several studies have suggested the potential benefits of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a precursor of heme, which generates reactive oxygen species (ROS) following photoirradiation. Some reports indicate that blue light induces intracellular ROS production. In the present study, we elucidated the effects of blue light and 5-ALA on DNA integrity in B16F1 murine melanoma and human keratinocyte HaCaT cells using a variety of comet assay techniques. Co-treatment with blue light and 5-ALA significantly decreased cell viability in both cell lines. A neutral comet assay was performed to assess DNA double-strand break (DSB) formation and blue light and 5-ALA caused DSBs. We also performed an alkali comet assay to detect single-strand breaks (SSB) and alkali labile sites (ALS). The results indicated that 5-ALA accelerated blue light-induced SSB formation. In addition, modified comet assays were done using two types of enzymes to evaluate oxidative DNA damages. The results indicated that blue light and 5-ALA generated oxidized purine and pyrimidines in both cell lines. In summary, co-treatment with 5-ALA and photoirradiation may cause unexpected DNA damage in cells and tissues.

Inhibition of estrogen receptor signaling as a strategy for radiosensitization of ER+ breast cancers

Purpose: The estrogen receptor (ER) is expressed in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of effective first-line therapies. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the effect of ET on tumor radiosensitization remains unclear, with concerns it may be radioprotective based on G1 cell arrest with ET treatment. Here we assessed the efficacy and mechanism of ER-mediated radiosensitization using various pharmacologic approaches in ER+ BC. Methods: Radiosensitization with ER inhibitors (tamoxifen [TAM], fulvestrant [FULV], AZD9496) was assessed using clonogenic survival assays. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) as well as changes in cell cycle, apoptosis, and senescence were assessed. The efficacy of TAM with RT in vivo was assessed with an MCF-7 xenograft model. Results: The selective estrogen receptor modulator TAM radiosensitized ER+ MCF-7 (enhancement ratio [enhR]: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (enhR: 0.99-1.02). The selective estrogen receptor degrader (SERD) FULV had similar radiosensitization effects in MCF-7 (enhR: 1.33-1.76) and T47D cells (enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (enhR: 1.01-1.03). The novel oral SERD AZD9496 radiosensitized MCF-7 cells (enhR: 1.36-1.56). MCF-7 cells treated with TAM and RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05) and a decrease in NHEJ-mediated repair with TAM (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or a reporter (p=NS). RT alone and in combination with TAM or FULV induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is cell-cycle independent. There were no significant changes in apoptosis with TAM, FULV, RT, or the combination (p=NS). Although TAM or FULV did induce senescence, ET with RT increased senescence induction (p<0.05). In vivo, combination RT and TAM led to a significant delay in days to tumor doubling (control: 17, TAM: 40, RT: 32, TAM+RT: undefined;p<0.0001), and a significant difference in tumor growth between mice treated with TAM or RT alone compared combination treatment, with no increased toxicities or skin lesions from the combination treatment. Conclusion: Our data suggest that TAM, FULV, or AZD9496 can radiosensitize ER+ breast tumors, and these agents with RT may be more effective for radiosensitization. This work also supports further clinical investigation of the timing of RT for patients receiving ET, including using ET during RT, especially as initiating ET prior to RT has been increasingly utilized as a bridging therapy followed by concurrent ET+RT during the COVID-19 pandemic.

Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers

Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of first-line therapies for ER-positive (ER+) BC patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remain unclear. Recently, this question has become much more clinically relevant when many physicians started offering ET as a bridging strategy to surgery and RT during the COVID-19 pandemic. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in preclinical models. Methods: Clonogenic survival assays were used to assess radiosensitization. Inhibition of ER signaling was accomplished by treating ER+ MCF-7 and T47D cells with the selective ER modulator (SERM), tamoxifen, or the selective ER degrader (SERD), fulvestrant. The ER-negative SUM-159 cells were used as a negative control. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) was measured by Rad51 foci or a GFP reporter system. Non-homologous end joining (NHEJ) efficiency was assessed with a pEYFP reporter. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow Scytometry. Senescence was measured using β-galactosidase staining. Western blotting was used to quantify expression of proteins and phospho-proteins involved in cell cycle and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Synergy was determined using the fractional tumor volume (FTV) method. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (10-250 nM, enhR: 1.14-1.50) and T47D (500 nM-2.0 μ M, enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (500 nM-2.0 μ M, enhR: 0.99-1.02). ER degradation with fulvestrant had similar radiosensitization effects in MCF-7 (1-25 nM, enhR: 1.33-1.76) and T47D cells (0.5-5 nM, enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (1-25 nM, enhR: 1.01-1.03). MCF-7 cells treated with 500 nM tamoxifen and 4 Gy RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05), and there was a decrease in NHEJ-mediated repair with tamoxifen treatment (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or an HR reporter (p=NS). RT alone and in combination with tamoxifen and fulvestrant induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is a cell-cycle independent effect. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with endocrine therapy, RT, or the combination (p=NS). Although treatment with ET did induce senescence in ER+ MCF-7 and T47D cells, the combination treatment of ET with RT induced senescence to a much greater level suggesting this mechanism may contribute to radiosensitization (p<0.05). In vivo, combination RT and tamoxifen led to a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen alone, 32 days with RT alone, and undefined with combination;p<0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment (FTV 1.297). Conclusion: Our data suggest that ET can radiosensitize ER+ breast tumors, and ET with RT may be more effective for radiosensitization. Ongoing studies will address concurrent versus sequential ET with RT. This work also supports further clinical investigation of the timing of RT for patients receiving ET, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID-19 pandemic.

Optimal multiparametric set-up modelled for best survival outcomes in palliative treatment of liver malignancies: unsupervised machine learning and 3 PM recommendations.

Over the last decade, a rapid rise in deaths due to liver disease has been observed especially amongst young people. Nowadays liver disease accounts for approximately 2 million deaths per year worldwide: 1 million due to complications of cirrhosis and 1 million due to viral hepatitis and hepatocellular carcinoma. Besides primary liver malignancies, almost all solid tumours are capable to spread metastases to the liver, in particular, gastrointestinal cancers, breast and genitourinary cancers, lung cancer, melanomas and sarcomas. A big portion of liver malignancies undergo palliative care. To this end, the paradigm of the palliative care in the liver cancer management is evolving from "just end of the life" care to careful evaluation of all aspects relevant for the survivorship. In the presented study, an evidence-based approach has been taken to target molecular pathways and subcellular components for modelling most optimal conditions with the longest survival rates for patients diagnosed with advanced liver malignancies who underwent palliative treatments. We developed an unsupervised machine learning (UML) approach to robustly identify patient subgroups based on estimated survival curves for each individual patient and each individual potential biomarker. UML using consensus hierarchical clustering of biomarker derived risk profiles resulted into 3 stable patient subgroups. There were no significant differences in age, gender, therapy, diagnosis or comorbidities across clusters. Survival times across clusters differed significantly. Furthermore, several of the biomarkers demonstrated highly significant pairwise differences between clusters after correction for multiple testing, namely, "comet assay" patterns of classes I, III, IV and expression rates of calgranulin A (S100), SOD2 and profilin-all measured ex vivo in circulating leucocytes. Considering worst, intermediate and best survival curves with regard to identified clusters and corresponding patterns of parameters measured, clear differences were found for "comet assay" and S100 expression patterns. In conclusion, multi-faceted cancer control within the palliative care of liver malignancies is crucial for improved disease outcomes including individualised patient profiling, predictive models and implementation of corresponding cost-effective risks mitigating measures detailed in the paper. The "proof-of-principle" model is presented.