Intravenous Infusion of High Dose Selenite in End-Stage Cancer Patients: Analysis of Systemic Exposure to Selenite and Seleno-Metabolites.

Cancer is one of the main causes of human death globally and novel chemotherapeutics are desperately required. As a simple selenium oxide, selenite is a very promising chemotherapeutic because of pronounced its dose-dependent tumor-specific cytotoxicity. We previously published a first-in-man systematic phase I clinical trial in patients with cancer (from IV to end-stage) (the SECAR trial) showing that selenite is safe and tolerable with an unexpectable high maximum tolerated dose (MTD) and short half-life. In the present study, we analyzed the selenium species in plasma samples, from the patients participating in the SECAR trial and from various time points and dose cohorts using LC-ICP-MS. In conclusion, selenite, selenosugars, and 1-2 unidentified peaks that did not correspond to any standard, herein denoted ui-selenium, were detected in the plasma. However, trimethylated selenium (trimethylselenonoium) was not detected. The unidentified ui-selenium was eluting close to the selenium-containing amino acids (selenomethionine and selenocysteine) but was not part of a protein fraction. Our data demonstrate that the major metabolite detected was selenosugar. Furthermore, the identification of selenite even long after the administration is remarkable and unexpected. The kinetic analysis did not support that dosing per the body surface area would reduce interindividual variability of the systemic exposure in terms of trough concentrations.

Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression.

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.

Soluble PD-L1 Expression After Intravenous Treatment of Cancer Patients With Selenite in Phase I Clinical Trial.

A high expression level of programmed death-ligand 1 (PD-L1) is observed in different types of cancers (particularly lung cancer). Soluble (s)PD-L1 may be used as a prognostic marker and a target for anti-cancer immunity, as well as, predicting gene therapy or systemic immunotherapy in blocking the PD-1 and PD-L1 checkpoint. Studies that evaluate the effects of the immune regulator selenium on PD-L1 expression show ambiguous results. Thus, we aimed to analyze sPD-L1 levels in samples from patients who underwent different dosages of selenite treatment in phase I clinical trial. We hypothesized that selenite modulates the sPD-L1 levels in the plasma as a consequence of the suggested mode of action of selenotherapy in cancer patients. In conclusion, our results support the view that selenotherapy does not substantially affect the PD-1/PD-L1 axis judged by sPD-L1 analysis. Furthermore, no significant correlation was observed between the survival and sPD-L1 expression nor sPD-L1 changes. However, due to a dynamic individual sPD-L1 profile and a high variation in survival, we suggest that further studies are needed to identify whether individual patients can be benefited from combinational seleno- and anti-PD-L1 therapy.

Selenoprotein P as Biomarker of Selenium Status in Clinical Trials with Therapeutic Dosages of Selenite.

Selenoprotein P (SELENOP) is an established biomarker of selenium (Se) status. Serum SELENOP becomes saturated with increasing Se intake, reaching maximal concentrations of 5-7 mg SELENOP/L at intakes of ca. 100-150 µg Se/d. A biomarker for higher Se intake is missing. We hypothesized that SELENOP may also reflect Se status in clinical applications of therapeutic dosages of selenite. To this end, blood samples from two supplementation studies employing intravenous application of selenite at dosages >1 mg/d were analyzed. Total Se was quantified by spectroscopy, and SELENOP by a validated ELISA. The high dosage selenite infusions increased SELENOP in parallel to elevated Se concentrations relatively fast to final values partly exceeding 10 mg SELENOP/L. Age or sex were not related to the SELENOP increase. Western blot analyses of SELENOP verified the results obtained by ELISA, and indicated an unchanged pattern of immunoreactive protein isoforms. We conclude that the saturation of SELENOP concentrations observed in prior studies with moderate Se dosages (<400 µg/d) may reflect an intermediate plateau of expression, rather than an absolute upper limit. Circulating SELENOP seems to be a suitable biomarker for therapeutic applications of selenite exceeding the recommended upper intake levels. Whether SELENOP is also capable of reflecting other supplemental selenocompounds in high dosage therapeutic applications remains to be investigated.

Expression profiles of thioredoxin family proteins in human lung cancer tissue: correlation with proliferation and differentiation.

AIMS: Lung cancer is one of the most common causes of cancer lethality worldwide. Despite recent progress, long-term survival remains poor. The aim of this study was to explore the expression pattern of the thioredoxin superfamily of proteins as potential new diagnostic and/or predictive markers. METHODS AND RESULTS: The expression of thioredoxin 1 (Trx1), thioredoxin reductase 1 (TrxR1), the isoforms TrxR1-v.2,3,5, glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2) was examined by immunohistochemistry on paraffin-embedded sections from 42 cases of non-small cell lung cancer patients. Additional cases of lung cancer from tissue microarray were examined and the immunoreactivity was compared. All proteins except TrxR1 showed a significant correlation with the degree of differentiation in adenocarcinoma. Trx1 and TrxR1-v.2,3,5 also showed a significant correlation with differentiation in squamous carcinoma. Furthermore, Grx1 and Grx2 showed a clear inverse correlation with proliferation. The proliferation rate was further analysed in vitro in stably transfected Grx2 overproducing cells, showing that the proliferative effect of Grx2 is strictly dependent on subcellular localization. CONCLUSIONS: The thioredoxin family of proteins is important for growth and differentiation of lung cancer cells. The correlation with differentiation and proliferation of these enzymes makes them promising predictive/diagnostic markers.

Comparison of predicted and clinical response to radiotherapy: a radiobiology modelling study.

INTRODUCTION: A model to predict clinical outcome after radiation therapy would be a valuable aid in the effort of developing more tailored treatment regimes for different patients. In this work we evaluate the clinical utility of a model that incorporates the following individually measured radiobiology parameters: intrinsic radiosensitivity, proliferation and number of clonogenic cells. The hypothesis underlying the study was that the incorporation of individually measured tumour parameters in a model would increase its reliability in predicting treatment outcome compared with the use of average population derived data. MATERIAL AND METHODS: Forty-six patients with head and neck tumours were analyzed, the majority of whom received both external beam radiotherapy and brachytherapy. Eighteen patients received external beam treatment alone and statistical analyses were carried out on this subgroup. RESULTS: Four of the 18 patients had a >95% calculated probability of cure and none developed a local recurrence resulting in a negative predictive value of 100% (compared with 67% for population-derived data). The sensitivity of the model in predicting local recurrence was 75% (compared with 38% for population-derived data). Using a model that incorporated individually measured radiobiology data, there was a statistically significant difference in local control levels for patients with >95% and <5% predicted probability of local control (chi(2), p = 0.04). DISCUSSION: This study suggests, therefore, that incorporation of measured biological data within a radiobiological model improves its ability to predict radiation therapy outcome compared with the use of population-derived data.

Pharmacokinetics and Toxicity of Sodium Selenite in the Treatment of Patients with Carcinoma in a Phase I Clinical Trial: The SECAR Study.

BACKGROUND: Sodium selenite at high dose exerts antitumor effects and increases efficacy of cytostatic drugs in multiple preclinical malignancy models. We assessed the safety and efficacy of intravenous administered sodium selenite in cancer patients' refractory to cytostatic drugs in a phase I trial. Patients received first line of chemotherapy following selenite treatment to investigate altered sensitivity to these drugs and preliminary assessment of any clinical benefits. MATERIALS AND METHODS: Thirty-four patients with different therapy resistant tumors received iv sodium selenite daily for consecutive five days either for two weeks or four weeks. Each cohort consisted of at least three patients who received the same daily dose of selenite throughout the whole treatment. If 0/3 patients had dose-limiting toxicities (DLTs), the study proceeded to the next dose-level. If 2/3 had DLT, the dose was considered too high and if 1/3 had DLT, three more patients were included. Dose-escalation continued until the maximum tolerated dose (MTD) was reached. MTD was defined as the highest dose-level on which 0/3 or 1/6 patients experienced DLT. The primary endpoint was safety, dose-limiting toxic effects and the MTD of sodium selenite. The secondary endpoint was primary response evaluation. RESULTS AND CONCLUSION: MTD was defined as 10.2 mg/m(2), with a calculated median plasma half-life of 18.25 h. The maximum plasma concentration of selenium from a single dose of selenite increased in a nonlinear pattern. The most common adverse events were fatigue, nausea, and cramps in fingers and legs. DLTs were acute, of short duration and reversible. Biomarkers for organ functions indicated no major systemic toxicity. In conclusion, sodium selenite is safe and tolerable when administered up to 10.2 mg/m(2) under current protocol. Further development of the study is underway to determine if prolonged infusions might be a more effective treatment strategy.

The role of circulating anti-p53 antibodies in patients with advanced non-small cell lung cancer and their correlation to clinical parameters and survival.

BACKGROUND: Lung cancer causes approximately one million deaths each year worldwide and protein p53 has been shown to be involved in the intricate processes regulating response to radiation and/or chemotherapeutic treatment. Consequently, since antibodies against p53 (anti-p53 antibodies) are associated with mutations within the p53 gene it seems likely that these antibodies could, hypothetically, be correlated with prognosis. METHODS: Serum samples from patients with non-small cell lung cancer (NSCLC) admitted to the Department of Oncology, University Hospital, Uppsala, Sweden, during 1983-1996 were studied. Anti-p53 abs were measured using a sandwich ELISA (Dianova, Hamburg, Germany). RESULTS: The present study included 84 patients with stage IIIA-IV (advanced NSCLC). At least three serum samples from each patient were collected and altogether 529 serum samples were analysed for the presence of anti-p53 antibodies. The median value of anti-p53 antibodies was 0.06 (range 0 - 139.8). Seventeen percent of investigated NSCLC first serum samples (n = 84) expressed elevated levels of anti-p53 antibodies. Anti-p53 antibodies were not correlated to tumour volume or platelets. Survival analysis showed that anti-p53 antibodies were not associated with survival as revealed by univariate analysis (p = 0.29). However, patients with adenocarcinoma had a significantly poorer survival if they expressed anti-p53 antibodies (p = 0.01), whereas this was not found for patients with squamous cell carcinoma (p = 0.13). In patients where the blood samples were collected during radiation therapy, a statistically significant correlation towards poorer survival was found (p = 0.05) when elevated anti-p53 antibodies levels were present. No correlations to survival were found for serum samples collected prior to radiation therapy, during chemotherapy, or during follow-up. When anti-p53 antibodies were measured continuously, no increase in median anti-p53 values was observed the closer the individual patient come to death. CONCLUSION: The result of the present retrospective study indicates that anti-p53 antibodies are not suitable for predictions concerning selection of patients with a more favourable outcome. Further prospective studies are, though, needed to fully elucidate this issue.

Tissue polypeptide antigen (TPA), hyaluronan and CA 125 as serum markers in malignant mesothelioma.

Mesothelioma is a rare disease with poor prognosis. Monitoring the effect of treatment is a problem and a serum marker might be of use for this purpose. We have studied three serum markers TPA, Hyaluronan and CA 125 in a limited material (11 patients) with the purpose of finding out if they might reflect treatment effect and/or indicate prognosis. The results in our material show that correspondence between initial TPA levels and survival seems to be better than corresponding data regarding Hyaluronan and CAI 25. Five patients show increasing serum levels of all three serum markers from first to last sample as the mesothelioma progressed according to consecutive CT scans. In three of these patients stable disease was followed by a decrease in the serum marker levels. Our results indicate that these three serum markers and mainly TPA might be useful as markers of disease progression and TPA for prediction of survival.

Radiation treatment of T1-T4 squamous cell carcinoma of the larynx: a retrospective analysis and long-term follow-up of 135 patients.

PURPOSE: To evaluate the long-term outcome of radiation therapy for patients with histologically verified squamous cell glottic carcinoma. PATIENTS AND METHODS: A retrospective study was performed on patients who had received radiation treatment at the Department of Oncology, Uppsala University, Uppsala, Sweden, between 1978 and 1987 Patients with a documented follow-up for at least five years, or to time of death, were studied. Radiation treatment was delivered utilising daily fractions of 1.8-20 over 6-7 weeks, totalling 60-70 Gy. Patients whose tumours were not controlled by radiation therapy, or whose tumours recurred, were offered surgical intervention. RESULTS: The study included 135 patients. Five-year survival rates adjusted to death due to laryngeal carcinoma were: T1, 95%; T2, 87%; T3, 72% and T4, 25%. CONCLUSION: Primary radiotherapy achieves a high rate of cure for T1-T2 laryngeal carcinoma. Tumour-related morbidity and death continued beyond the standard five-year follow-up, especially for patients with T1/T2 laryngeal carcinomas.

p53 status and its in vitro relationship to radiosensitivity and chemosensitivity in lung cancer.

The following study was designed to investigate if mutations within the p53 gene are associated with radiation responsiveness or response to different cytotoxic drugs. Nine human lung cancer cell lines were examined (four SCLC and five NSCLC cell lines). cDNA-based sequencing of the entire p53 gene was performed. All cell lines were characterised with respect to drug-sensitivity towards eight cytotoxic drugs using the FMCA method and data from the clonogenic assay were studied to obtain information concerning radioresponsiveness. All the cell lines expressed mutations; six were missense mutations and three were deletions. A statistically significant increase in radiosensitivity was found for mutations in exon 7 (p = 0.019), compared with the other mutations localised within different exons of p53. Further statistical analyses using Fisher's two-tailed exact test confirmed that mutations in exon 7 were significantly associated with radiosensitivity, p = 0.047. No correlation concerning mutations in separate exons and response towards different chemotherapeutic agents could be found. Our results indicate that p53 mutations in exon 7 might be associated with increased radiation sensitivity in these human lung cancer cell lines, but our data should be interpreted with caution since several other explanations might exist regarding what determines the response towards radiation.

Selection of chemotherapy by ex vivo assessment of tumor sensitivity to cytotoxic drugs: results of a clinical trial.

The feasibility of tumor sampling followed by ex vivo assessment of drug sensitivity, using the short-term fluorometric microculture cytotoxicty assay (FMCA), for selection of chemotherapy was investigated prospectively in patients with advanced cancer not amenable to standard treatment. Taxol (175 mg/m2 every 3 wk) was given to patients with tumor samples being low drug resistant (LDR) to Taxol ex vivo, to patients with no LDR drug, and if other drugs were unsuitable. The remaining patients received the most optimal drug(s) based on the FMCA results. Gastrointestinal cancer was dominating among the 61 eligible patients. Tumor sampling was safely performed in 75% by ultrasound-guided core biopsy. Eighty-two percent of the patients had Taxol. Five patients (8%) had a partial remission and 18 (30%) had stable disease. Tumor response was poorly predicted, probably because the Taxol excipient Cremophor EL is cytotoxic exclusively ex vivo. However, patients with tumor cells being LDR to at least one drug ex vivo lived significantly longer than those with no such drug.

The value of induction chemotherapy for survival in patients with non-small cell lung cancer treated with radiotherapy.

AIM: The aim of the present study was to retrospectively investigate the impact of induction chemotherapy on treatment outcome in patients treated with curatively intended radiotherapy for non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with a diagnosed NSCLC that have been subjected to curatively intended irradiation (≥50 Gy) and treated in an oncology department in Sweden during the years 1990-2000 were included in the study. Operated patients and patients having received concomitant chemotherapy were excluded. The included patients were localised by a manual search of all the oncology departments' medical records and radiation charts. RESULTS: Patients treated with induction chemotherapy (n=79) had a significantly better overall survival compared with patients treated with radiotherapy alone (p=0.0097) in a univariate Cox regression analysis. A platinum/taxane combination produced the greatest survival benefit; hazard ratio=0.49 (95% confidence interval=0.31 to 0.75). CONCLUSION: We found that patients treated with induction chemotherapy in addition to radiotherapy for NSCLC have a better overall survival than patients treated with radiotherapy alone and that the best results are achieved using a platinum/taxane combination.

Fractionated irradiation of five human lung cancer cell lines and prediction of survival according to a radiobiology model.

BACKGROUND: This study evaluates a predictive radiobiology model by measurements of surviving fraction (SF) by the clonogenic assay or the extrapolation method and the proliferation rate in vitro. It is hypothesized that incorporating proliferation to intrinsic radiosensitivity, measured by SF, to predict radiation responsiveness after fractionated irradiation adds to the model's accuracy. Materials and Methods. Five lung cancer cell lines with known SF after 1 Gy (SF1), and also SF2 and SF5, were irradiated with three different fractionation regimes; 10 × 1 Gy, 5 × 2 Gy or 2 × 5 Gy during the same total time to achieve empirical SF. In addition, the SF1, SF2 and SF5 after fractionated irradiation was calculated for each cell line based on the already known single fraction SF and with or without a proliferation factor. The results were compared to the empirical data. RESULTS AND DISCUSSION: By using the clonogenic assay to measure radiosensitivity, prediction of radiosensitivity was improved after fractionated radiotherapy when proliferation was used in the radiobiology model. However, this was not the case in the cell lines where the extrapolation method was used to calculate SF. Thus, a radiobiology model including intrinsic radiosensitivity, measured by the clonogenic assay, as well as proliferation, is better at predicting survival after fractionated radiotherapy, compared to the use of intrinsic radiosensitivity alone.

Treatment of lung cancer cells with cytotoxic levels of sodium selenite: effects on the thioredoxin system.

Selenium at subtoxic doses has been shown to have tumor specific cytotoxic effects. In this work, viability measurements in different lung cancer cell lines showed that selenite was more effective compared to three different conventional cytotoxic drugs. In addition, the cell line most sensitive to selenite toxicity comprised the highest level of thioredoxin reductase 1 (TrxR1). The human selenoenzyme TrxR1 is a central enzyme for cell growth, differentiation, and the protection against oxidative stress. TrxR1, which in several studies has been shown to be up-regulated in various tumor cells, is also a target for many anticancer drugs. In this study, inhibition of TrxR resulted in enhanced selenite cytotoxicity, clearly connecting the thioredoxin system to the toxic effects mediated by selenite. The complex regulation of TrxR1, involving the expression of many different transcript forms of mRNA, was investigated by real-time qPCR in lung cancer cell lines following treatment with toxic doses (2.5-10 microM) of sodium selenite. Selenium treatment resulted in increased expression of almost all TrxR1 mRNA variants with increasing concentrations of selenite. On the contrary, the TrxR protein level and activity, increased at low to moderate doses followed by a decrease at higher doses, indicating impairment of protein synthesis by selenite.

Endothelial markers and circulating angiogenic factors and p53 may be potential markers for recurrence in surgically resected non-small cell lung cancer patients.

BACKGROUND: Early stage lung cancer is potentially curable by resection, but 30-50% of patients will relapse within five years after surgery. Therefore, the search for a predictive method capable of estimating the risk of recurrence in this population of patients is important. MATERIAL/METHODS: We analysed, on the one hand, the predictive powers for recurrent disease of the immunohistochemical expressions of p53 and the endothelial markers CD34 and CD105 in 53 NSCLC tumor samples, and, on the other hand, their correlations to serum VEGF and bFGF levels. Moreover, we sequenced the whole coding region of the p53 gene in 32 tumor samples for the presence of p53 mutations (exons 2-11) using a cDNA technique. RESULTS: The two endothelial markers correlated with each other. CD105 expression correlated with p53 expression, which was overexpressed in 49%. No other significant correlations between markers could be demonstrated. A significant correlation between p53 overexpression and recurrent disease was demonstrated (p=0.029). The mutational status of p53 correlated to p53 protein overexpression, but did not correlate with either of the immunohistochemical markers. The mutational status could not confirm an immunohistochemical correlation between p53 and recurrences (p=0.068). CONCLUSIONS: The present study demonstrates that p53 expression correlates with CD105 expression, and that p53 overexpression may indicate a lower recurrence risk in patients undergoing surgery for NSCLC stage I-IIIA, although future larger prospective studies are needed to fully elucidate this finding.

HER-2, EGFR, COX-2 expression status correlated to microvessel density and survival in resected non-small cell lung cancer.

The incidence of lung cancer is increasing throughout the world and is the most common cause of cancer-related death. Early detection followed by surgery has a reasonable, curative potential, but 30-50% of patients experience relapses. The immunohistochemical expressions of HER-2, EGFR and COX-2 were investigated in 53 resected non-small cell lung carcinomas and correlated to microvessel density and clinical data. HER-2, EGFR and COX-2 overexpressions were demonstrated in 15%, 30% and 40% of the tumours, respectively. In adenocarcinomas, HER-2 and COX-2 overexpression were more common, whereas in squamous cell carcinomas, EGFR overexpression was more common. COX-2 expression correlated with HER-2 expression (p = 0.002), and demonstrated a trend towards a correlation with microvessel density (p = 0.10). None of the markers alone had any impact on survival. However, HER-2+/EGFR- tumours proved to have a poor prognosis. In conclusion, adjuvant treatment with HER-2 antagonists might be a future treatment option in resected non-small cell lung cancer patients, especially when HER-2 is overexpressed without a concomitant overexpression of EGFR.