Etoposide Concentrations Research Articles

Label-free high temporal resolution assessment of cell proliferation using digital holographic microscopy.

Cell proliferation assays are widely applied in biological sciences to understand the effect of drugs over time. However, current methods often assess cell population growth indirectly, that is, the cells are not actually counted. Instead other parameters, for example, the amount of protein, are determined. These methods often also demand phototoxic labels, have low temporal resolution, or employ end-point assays, and frequently are labor intensive. We have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using digital holographic microscopy (DHM), one of many quantitative phase microscopy techniques. As no labels or stains are required, and only very low intensity illumination is necessary, the technique allows for noninvasive continuous cell counting. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. The developed direct assay was validated by analyzing cell cultures treated with various concentrations of the anti-cancer drug etoposide, a well-established topoisomerase inhibitor that causes DNA damage and leads to programmed cell death. After treatment, the unstained adherent cells were nondestructively imaged every 30 min for 36 h inside a cell incubator. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. Our results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods. © 2017 International Society for Advancement of Cytometry.

Genomic analysis of drug resistant small cell lung cancer cell lines by combining mRNA and miRNA expression profiling.

Etoposide (VP16) combined with cisplatin (DDP), as the first-line chemotherapy for small cell lung cancer (SCLC), regularly confers drug resistance. The present study applied complementary (c)DNA and micro (mi)RNA microarray to identify gene and miRNA expression profiles associated with multidrug resistance (MDR) in SCLC. The VP16/DDP (VP16 combined with DDP) resistant SCLC H446/EP cell line was derived from the parental H446 cell line by continuous exposure to increasing concentrations of etoposide and cisplatin. The mRNA and miRNA expression profiles between the resistant and parental SCLC cells were analyzed by Phalanx OneArray™ mRNA and miRNA microarray, and the results were confirmed by quantitative polymerase chain reaction. The expression levels of 75 genes were downregulated whilst 40 genes were upregulated in the H446/EP cell line compared with the H446 cell line. The expression levels of 16 miRNAs were upregulated whilst 15 were downregulated in the H446/EP cell line compared with the H446 cell line. Expression profile studies indicate that the particular mRNA and miRNA alteration demonstrated in MDR of SCLC may provide potential biomolecular targets for MDR reversion.

Supervised classification of etoposide-treated in vitro adherent cells based on noninvasive imaging morphology.

Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for [Formula: see text] etoposide, whereas effects on MTS and viability are detected only on day 3 for [Formula: see text] etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we report a best case sensitivity of 88% and specificity of 94% for classification of cells as treated/untreated.

Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage.

A quantitative dynamics pathway map of the Nrf2-mediated oxidative stress response and p53-related DNA damage response pathways as well as the cross-talk between these pathways has not systematically been defined. To allow the dynamic single cell evaluation of these pathways, we have used BAC-GFP recombineering to tag for each pathway's three key components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP, and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and p21-GFP. The dynamic activation of these individual components was assessed using quantitative high throughput confocal microscopy after treatment with a broad concentration range of diethyl maleate (DEM; to induce oxidative stress) and etoposide (to induce DNA damage). DEM caused a rapid activation of Nrf2, which returned to baseline levels at low concentrations but remained sustained at high concentrations. Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes followed later, with upper boundaries reached at high concentrations, close to the onset of cell death. Etoposide caused rapid accumulation of 53bp1-GFP in DNA damage foci, which was later followed by the concentration dependent nuclear accumulation of p53-GFP and subsequent induction of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest activation of DNA damage reporters was observed for DEM at high concentrations. Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage response at high concentrations of etoposide, while Keap1 knockdown caused an enhancement of the DNA damage response already at low concentrations of etoposide. Knockdown of p53 did not affect the oxidative stress response. Altogether, the current stress response landscapes provide insight in the time course responses of and cross-talk between oxidative stress and DNA-damage and defines the tipping points where cell injury may switch from adaptation to injury.

Pharmacokinetics and dose adjustment of etoposide administered in a medium-dose etoposide, cyclophosphamide and total body irradiation regimen before allogeneic hematopoietic stem cell transplantation.

BackgroundWe investigated the pharmacokinetics of etoposide (ETP) to reduce the inter-individual variations of ETP concentrations in patients with acute leukemia who underwent allogeneic hematopoietic stem cell transplantation. We also carried out an in vivo study using rats to verify the dose adjustment.MethodsThis study included 20 adult patients. ETP was administered intravenously at a dose of 15 mg/kg once daily for 2 days (total dose: 30 mg/kg) combined with standard conditioning of cyclophosphamide and total body irradiation. In an in vivo study using rats, ETP was administered intravenously at a dose of 15 mg/kg or an adjusted dose. The ETP plasma concentration was determined by using HPLC. The pharmacokinetic parameters were estimated by using a 1-compartment model.ResultsThe peak concentration (Cmax) of ETP and the area under the plasma concentration-time curve (AUC) of ETP differed greatly among patients (range of Cmax, 51.8 - 116.5 μg/mL; range of AUC, 870 - 2015 μg · h/mL). A significant relationship was found between Cmax and AUC (R = 0.85, P < 0.05). Distribution volume (Vd) was suggested to be one of the factors of inter-individual variation in plasma concentration of ETP in patients (range of Vd, 0.13 - 0.27 L/kg), and correlated with Alb and body weight (R = 0.56, P < 0.05; R = 0.40, P < 0.05 respectively). We predicted Vd of rats by body weight of rats (with normal albumin levels and renal function), and the dose of ETP was adjusted using predicted Vd. In the dose adjustment group, the target plasma ETP concentration was achieved and the variation of plasma ETP concentration was decreased.ConclusionThe results suggested that inter-individual variation of plasma concentration of ETP could be reduced by predicting Vd. Prediction of Vd is effective for reducing individual variation of ETP concentration and might enable a good therapeutic effect to be achieved.

Glutamine deprivation plus BPTES alters etoposide- and cisplatin-induced apoptosis in triple negative breast cancer cells.

Glutamine provides cancer cells with the energy required to synthesize macromolecules. Methods which block glutamine metabolism in treatment of breast cancer inhibit oncogenic transformation and tumor growth. We investigated whether inhibiting glutamine metabolism produces effects that are synergistic with those produced by drugs which damage DNA in triple-negative breast cancer cells. HCC1937 and BT-549 breast cancer cells were co-treated with either cisplatin or etoposide in combination with BPTES (a specific inhibitor of glutaminase 1) or exposure to a glutamine-free medium, and the cell proliferation and cell apoptosis were measured by flow cytometry, immunoblotting studies, and CCK-8 assays. The results showed that both glutamine deprivation and BPTES pretreatments increased the toxic effects of cisplatin and etoposide on HCC1937 cells, as demonstrated by their reduced proliferation, increased expression of apoptosis-related proteins (cleaved-PARP, cleaved-caspase 9, and cleaved-caspase 3) and decreased Bcl-2/BAX ratio. However, in BT-549 cells, glutamine deprivation and BPTES treatment increased etoposide-induced apoptosis only when used with higher concentrations of etoposide, and the effect on cisplatin-induced apoptosis was minimal. These results suggest that the anti-cancer effects produced by a combined approach of inhibiting glutamine metabolism and administering common chemotherapeutic agents correlate with the tumor cell type and specific drugs being administered.

Population Pharmacokinetic Modeling of Etoposide Free Concentrations in Solid Tumor.

This study aimed to determine free etoposide (ETO) concentrations in two regions of Walker-256 (W256) solid tumor using microdialysis and to establish a population pharmacokinetic (popPK) model to describe simultaneously free tumor and total plasma concentrations. W256 tumor-bearing Wistar rats received ETO 10 or 20mg/kg i.v. bolus. Free ETO concentrations were sampled from central and peripheral regions of the tumor via CMA/20 probes for up to 7h, whereas blood samples were collected via carotid artery cannulation. Total plasma and free tumor concentration-time profiles were analyzed by non-compartmental approach using WinNonlin® v. 5.3. PopPK modeling was conducted using MONOLIX v.4.3.3. ETO penetration was higher in the periphery (61 ± 15% and 61 ± 29%) than in tumor center (34 ± 6% and 28 ± 11%) following 10 and 20mg/kg doses, respectively (ANOVA, α = 0.05). A 4-compartment model fitted ETO concentration-time profiles in all sampling compartments. The popPK model allowed the simultaneous fitting of plasma and tumor concentrations and a better understanding of ETO distribution in solid tumors. ETO plasma concentrations are not a good surrogate for tumoral exposure, emphasizing the importance of knowing intratumoral concentrations to predict drug response.

Micronization of etoposide using solution-enhanced dispersion by supercritical CO2

To improve the solubility and dissolution rate of etoposide, micro-particles were prepared using solution-enhanced dispersion by supercritical CO2, and the transition of crystal form was investigated using scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and differential scanning calorimetry. The effects of various operating parameters, including precipitation pressure, temperature, the etoposide concentration, and solution flow rate were investigated. The results showed that the particle size of etoposide can be controlled, and smaller particles of 586nm were successively obtained. A reduction in the particle size led to an increase in the dissolution rate. Furthermore, at a precipitation pressure ≥18MPa, a new polymorphic form was discovered, which exhibited higher solubility and faster dissolution rate.

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL−1, respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

Adaptive dosing of anticancer drugs in neonates: facilitating evidence-based dosing regimens.

PurposeSelection of the most appropriate chemotherapy dosing regimens for neonates treated within the first weeks of life represents a significant clinical dilemma. Due to a lack of information relating to the clinical pharmacology of anticancer drugs in these challenging patients, current dosing guidelines are based on limited scientific rationale. In the current study, we investigate the utilisation of therapeutic drug monitoring approaches in neonates with localised hepatoblastoma, Wilms’ tumour and stage 4S neuroblastoma, being treated with widely used anticancer drugs.MethodsPlasma concentrations of cisplatin, vincristine, etoposide and carboplatin were quantified in two neonates being treated within the first 3 weeks of life and in a 32-week preterm infant treated at a gestational age of 40 weeks. Therapeutic drug monitoring was carried out where appropriate, based on the pharmacokinetic data obtained in conjunction with clinical response and toxicity.ResultsTreatment of a child aged 2 weeks with a recommended cisplatin dose reduction for weight to 1.8 mg/kg resulted in achievement of unbound cisplatin plasma concentrations of 0.01–0.08 µg/mL, markedly lower than exposures previously reported in infants and older children. A dose increase to 2.7 mg/kg was implemented, leading to the achievement of levels more in-line with those previously reported. This increased dose level was well tolerated over six courses of treatment, resulting in a good response to cisplatin monotherapy and the patient remains in remission at 3.5 years. In contrast, a 50 % vincristine dose reduction for weight in a 3-week-old neonate resulted in plasma concentrations comparable to levels observed in older children, leading to successful treatment and continued remission at 2 years. In a third patient, etoposide and carboplatin clearance values normalised to body weight were comparable to those reported in older children, resulting in comparatively lower exposures following reduced dosing.ConclusionsThe current report provides unique data on the pharmacokinetics of several widely used anticancer drugs in neonates treated within the first few weeks of life. The provision of these data acts as a useful reference point to support future dosing decisions to be made by clinicians in the treatment of these challenging patients.

Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species.

Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34+ cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34+ cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid leukemia cells, and it is harmless to normal human cells.

A simple dried blood spot method for clinical pharmacological analyses of etoposide in cancer patients using liquid chromatography and fluorescence detection

BackgroundTherapeutic drug monitoring of etoposide is not part of the routine clinical practice, however, measuring etoposide plasma concentration may be useful to prevent chemotherapy related adverse drug reactions. This paper describes the development and validation of a dried blood spot (DBS) assay for the determination of etoposide in blood samples of lung cancer patients. MethodsThe whole blood spot was cut out of the DBS card followed by sonication assisted liquid drug extraction. Extraction solution was evaporated and re-dissolved. A high-performance-liquid-chromatography method with fluorimetric detection ( λex=230nm; λem=330nm) was used. ResultsMethod met the validation criteria in terms of selectivity, linearity (0.5–20.0μg/mL), accuracy (≥96.1%), precision (≤10.1%) and stability (long term 4weeks at room temperature and 40°C). Haematocrit did not influence DBS etoposide concentration. Good correlation between measured plasma and DBS concentrations was observed. The equation considering only haematocrit value was used for conversion of DBS to plasma concentration. ConclusionsDBS sampling method showed comparable results to plasma samples. Therefore, it can be concluded that the developed and validated DBS method, which is more patient-friendly and requires less sample handling, is a reliable alternative to conventional plasma methods for measuring etoposide concentration in clinical pharmacological analyses.

Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells.

Etoposide induces cell death via mitochondrial-dependent actions of p53.

BackgroundEtoposide has been used clinically in cancer treatment, as well as in numerous research studies, for many years. However, there is incomplete information about its exact mechanism of action in induction of cell death.MethodsEtoposide was compared at various concentrations to characterize the mechanisms by which it induces cell death. We investigated its effects on mouse embryonic fibroblasts (MEFs) and focused on both transcriptional and non-transcriptional responses of p53.ResultsHere we demonstrate that treatment of MEFs with higher concentrations of etoposide induce apoptosis and activate the transcription-dependent functions of p53. Interestingly, lower concentrations of etoposide also induced apoptosis, but without any evidence of p53-dependent transcription up-regulation. Treatment of MEFs with an inhibitor of p53, Pifithrin-α, blocked p53-dependent transcription but failed to rescue the cells from etoposide-induced apoptosis. Treatment with PES, which inhibits the mitochondrial arm of the p53 pathway inhibited etoposide-induced cell death at all concentrations tested.ConclusionsWe have demonstrated that transcriptional functions of p53 are dispensable for etoposide-induced cell death. The more recently characterized effects of p53 at the mitochondria, likely involving its interactions with BCL-2 family members, are thus more important for etoposide’s actions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0231-z) contains supplementary material, which is available to authorized users.

Inhibition of topoisomerase IIα sensitizes FaDu cells to ionizing radiation by diminishing DNA repair.

Despite the high efficiency of ionizing radiation (IR) to inactivate malignant tumours in general, an appreciable number of individual patients cannot be cured by standard IR. Head and neck tumours are not likely to be cured even by high-dose radiotherapy or chemotherapy. Accordingly, combined therapy is one of the most applicable strategies. Topoisomerase IIα is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating and subsequently resealing of transient double-strand breaks. Due to its unique mechanism of action, topoisomerase IIα is the target of many chemotherapeutic agents such as etoposide. The aim of the present study is to examine the effect of inhibiting topoisomerase IIα by etoposide on the response of squamous cell carcinoma to IR. Results of the present study demonstrated a radiosensitizing effect for the topoisomerase IIα inhibitor etoposide on exponentially growing squamous cell carcinoma (FaDu) cell line especially at low radiation doses. This effect was found to be due to inhibition, by etoposide, of the repair of radiation-induced DNA damage. Cell cycle studies showed that the concentration of etoposide that sensitized the cells to radiation had no effect on the distribution of cells at different phases of the cell cycle. Synchronization of FaDu cells in different cell cycle phases revealed that proliferating G1 and G2 cells are responsible for sensitization of cells at low doses of ionizing radiation. It might, therefore, be concluded that topoisomerase II enzyme may be involved in the repair of radiation-induced DNA damage and consequently its inhibition constitute a strategy for sensitizing tumour cells to ionizing radiation.

A pilot study of bevacizumab combined with etoposide and cisplatin in breast cancer patients with leptomeningeal carcinomatosis

BackgroundElevated vascular endothelial growth factor (VEGF) was associated with poor prognosis in leptomeningeal carcinomatosis and anti-angiogenic therapy was found to prolong the survival of mice in preclinical studies. This prospective pilot study investigated the efficacy of anti-VEGF therapy plus chemotherapy in patients with leptomeningeal carcinomatosis originating from breast cancer.MethodsEligible patients were scheduled to receive bevacizumab combined with etoposide and cisplatin (BEEP) every 3 weeks for a maximum of 6 cycles or until unacceptable toxicity. The primary objective was the central nervous system (CNS)-specific response rate, which was defined as disappearance of cancer cells in the cerebrospinal fluid (CSF) and an improved or stabilized neurologic status. The impact of VEGF inhibition on etoposide penetration into the CSF was analyzed.ResultsEight patients were enrolled. The CNS-specific response rate was 60% in 5 evaluable patients. According to intent-to-treat analysis, the median overall survival of the eight patients was 4.7 months (95% confidence interval, CI, 0.3–9.0) and the neurologic progression-free survival was 4.7 months (95% CI 0–10.5). The most common grade 3/4 adverse events were neutropenia (23.1%), leukopenia (23.1%), and hyponatremia (23.1%). The etoposide concentrations in the CSF were much lower than those in plasma, and bevacizumab did not increase etoposide delivery to the CSF.ConclusionsBEEP exhibited promising efficacy in breast cancer patients with leptomeningeal carcinomatosis. Additional studies are warranted to verify its efficacy and clarify the role of anti-angiogenic therapy in this disease.Trial registrationClinicalTrials.gov identifying number NCT01281696.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1290-1) contains supplementary material, which is available to authorized users.

BCL6 Dose Not Negatively Regulate DNA Damage Response Genes in Multiple Myeloma Cells

Background: A proto-oncogene BCL6 is a known transcriptional repressor and a master regulator of germinal center B cell program. It represses expression of DNA damage response genessuch as p53, ATR, CHEK1 and p21, which helps B cells to survive and expand during antigen receptor-diversification reactions. It also plays a pivotal role in the formation of germinal centers and lymphomagenesis. BCL6 is down-regulated in normal plasma cells while it is aberrantly expressed in bone marrow residing myeloma cells. Although the role of BCL6 in B cell lymphomas has been intensively studied, its role in plasma cell neoplasms remains to be elucidated. In this study we asked whether BCL6 plays a role in DNA damage response of myeloma cells.Methods: Bone marrow samples obtained from 36 of primary plasma cell dyscrasia patients (5 cases of MGUS, 30 of multiple myeloma (MM) and 1 plasma cell leukemia (PCL)) were subjected to the study after informed consent. This study was approved by IRBs of Gunma University Hospital and Nishigunma National Hospital. CD138-positive bone marrow plasma cells were isolated as a purity of >95% using magnetic beads and RNAs were extracted. Expression levels of BCL6, p53, ATR, CHEK1 and p21 were quantified by real time PCR using Taqman-probes. For retroviral infection, BCL6 was cloned into pMY-IRES-GFP vector and transfected to PlatA cells using lipofection reagents. 48 hours later, supernatants were collected and centrifuged for 16 hours and the pellets were used for infection. GFP positive cells were collected and used for following experiments. For γH2AX foci formation analysis, the cells were given ionized irradiation at doses of 0, 3, 5 and 10Gy, and used after an hour incubation. Cells were also exposed to different concentration of etoposide (0, 1, 5, 10, 50, 100μM) for 30minute, then washed with fresh media and incubated for an hour. Cells were stained with a FITC-labeled anti-γH2AX antibody as reported (Muslimovic et al, Nat. Protoc. 2008). For cell cycle analysis by flow cytometry, EdU uptake and 7AAD DNA staining were performed according to a manufacture’s protocol (Life Technologies). Correlation of expression levels between each of genes was assessed using Spearman’s non-parametric correlation analysis.Results: Median of BCL6 expression levels of MM and PCL cells (Qty median=1.47, range 0.09-17.2) was not significantly different from that of MGUS cells (Qty median=1.84, range 0.8-4.2, p=0.51). In marked contrast to germinal center B cells, expression levels of BCL6 and p53 were positively correlated in MM, PCL and MGUS (r=0.457, p=0.007). The correlation between expression of BCL6 and ATR did not reach statistical significance (r=0.323, p=0.062). ATR and p53 were also positively correlated (r=0.549, p=0.001). The expression level of p21, a well-known target of p53, was positively correlated to that of p53 (r=0.681, p=0.03), which supports our data qualification. We also examined mRNA expression levels of BCL6 in MM cell lines, RPMI8226, KMS11, KMS12PE, KMS12BM, KMS18, KMS26, ARH 77 and U266. U266, KMS12PE, KMS26 expressed little amount of BCL6 compared to the patient samples. The other five cell lines did not express BCL6. In order to study BCL6 functions in MM cells, we retrovirally expressed BCL6 in the KMS12BM cell line. The expression level of BCL6 was comparable to the patient samples after the retroviral expression (QTy 6.70). Since BCL6 is known to be a transcriptional repressor and supposed to directly repress p53, ATR, CHEK1 and p21 in B cells, we analyzed expression levels of these genes. Intriguingly, p53, ATR, CHEK1 and p21 are not repressed by overexpression of BCL6 in KMS12BM. These results suggest that alternative regulatory mechanisms of transcriptional regulation by BCL6 are present in MM cells. For further evaluation of the DNA damage response by BCL6 expression, we irradiated or treated these cells with etoposide and analyzed for γH2AX foci formation, a hallmark of DNA double strand break. No differences in the foci formation between mock-infected and BCL6-infected-KSM12BM were detected either with irradiation or etoposide exposure. We also studied cell cycle progressionin these cells. Flow cytometry analysis showed no significant difference between these cells.Conclusion: Unlike B cells in germinal centers, BCL6 expression in myeloma cells did not repress DNA damage response gene expression. DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics and Tolerability of Etoposide in Newly Diagnosed Lymphoma Patients with Hepatic Impairment

Background: The addition of etoposide to chemotherapy regimens (e.g.- CHOEP and EPOCH) may be beneficial for the treatment of aggressive lymphomas. Previous studies have shown decreased clearance of etoposide in hepatic impairment leading to a dose reduction or removal of drug. Total clearance of etoposide does not change significantly in patients with elevated bilirubin. However, free (or unbound) etoposide levels are a more accurate measurement of drug clearance (Stewart el al. Changes in the Clearance of Total and Unbound Etoposide in Patients with Liver Dysfunction. J Clin Oncol. 1990 Nov;8(11):1874-9.). No studies have analyzed etoposide pharmacokinetics and clinical toxicity in lymphoma patients with hepatic impairment.Methods: Patients with newly diagnosed aggressive lymphoma received 1 to 8 cycles of DA-EPOCH +/-R at the NCI. Of 56 pts studied, 48 had normal and 8 had elevated bilirubin (median 3.4mg/dL, range 1.4-22.5mg/dL). All patients received full dose etoposide at 200mg/m2 over 96-hours on cycle 1. Blood samples were collected at 0, 22 and 96 hours after infusion began.Results: There was no significant difference between free etoposide clearance (Cl) and the free etoposide concentration (Css) in patients with elevated versus normal bilirubin (see table below). In all patients, irrespective of bilirubin, there was no significant correlation between free etoposide Cl, free etoposide Css and creatinine clearance (CrCl) or age. Neutropenia and thrombocytopenia was higher in patients with elevated bilirubin. Complete response (CR) was achieved in 75% (6/8) versus 85% (41/48) and PR in 0% (0/8) vs 4% (2/48) of patients with elevated vs normal bilirubin.Conclusions: Etoposide pharmacokinetics were not altered in patients with abnormal versus normal hepatic function. Although there was an increase in hematologic toxicities, there was no difference in Css or Cl of etoposide. Importantly, there were no significant differences in febrile neutropenia or grade 3/4 toxicities. The toxicity difference is likely attributed to other drugs. Response rates were similar between the 2 groups. Our results do not support the empiric dose reduction of etoposide in patients with aggressive lymphomas receiving potentially curative treatment.TableResultsElevated BilirubinNormal Bilirubinp-valueMedian age (yrs)39 (17-59)54 (20-75)0.15 IPI (int hi/hi risk)75% (6/8)23% (11/48)0.0070ANC nadir < 500/mm3 (C1)100% (8/8) 54% (26/48)0.017PLT nadir < 25x103/mm3 (C1)38% (3/8)4% (2/48)0.017Febrile Neutropenia (C1)13% (1/8)13% (6/48)1.00G3/4 GI and neuro tox (C1)38% (3/8)13% (6/48)0.11Median free etoposide clearance on C1 (ml/min/m2)724 (438-1413)663 (199-1148)0.40Median free etoposide concentration on C1 (μM)0.082 (0.044-0.135)0.089 (0.052-0.298)0.44 DisclosuresNo relevant conflicts of interest to declare.

OP27 * ETOPOSIDE PHARMACOKINETICS FOLLOWING INTRA-CEREBROSPINAL FLUID ADMINISTRATION IN PATIENTS WITH LEPTOMENINGEAL METASTASES

INTRODUCTION: Daily intra-cerebrospinal fluid (CSF) etoposide administration regimens are in use, by-passing the blood-brain-barrier and offering higher CSF drug concentrations at 1/30th to 1/340th the dose in conventional systemically administered standard and high dose etoposide regimens, respectively. METHOD: 42 pharmacokinetic samples were obtained from a paediatric (36) and adult patient (6). Etoposide 0.75 mg was administered over 1-2 minutes via the intra-ventricular (IVT) or lumbar route (LR), on 5 consecutive days for 1-3 weeks. CSF sampling at 4h (IVT: 3, LR: 3), 8h (IVT: 3, LR: 2), 18h (IVT: 1, LR: 1), 24h (IVT: 11, LR: 9), and 72h (IVT: 5, LR: 4). Etoposide concentrations were determined by LC/MS analysis, with a detection limit of 0.1 µg/ml. RESULTS: No acute neurotoxicity was observed. Single IVT administration with LR sampling yielded higher concentrations than LR administration with IVT sampling at 4h and 8h (1.73 and 0.93 µg/ml, vs. 1.42 and 0.76 µg/ml, respectively), also observed with simultaneous LR and IVT administration (median LR concentration 5.11 and 1.77 µg/ml at 4h and 8h; median IVT concentration 3.64 and 1.48 µg/ml at 4h and 8h). Concentrations at 24h were <0.2 (median 0.15, 14 samples) and at 72h <0.1 µg/ml (8 samples), irrespective of routes and sample sites. CONCLUSION: Intra-CSF etoposide administration at this dose level is safe, with almost complete clearence at 24h, and good distribution throughout the CSF compartment, irrespective of delivery route. Higher LR compared to IVT concentrations are probably related to CSF stasis. The rapid clearance characteristic warrants investigation of continuous drug infusion to sustain targeted CSF steady state etoposide concentrations.

Pharmacokinetics (Pk) of Oral Etoposide (Oe) in Kenyans with Hiv-Related Kaposi'S Sarcoma (Ks) on Anti-Retroviral Therapy (Art)

ABSTRACT Aim: Recent trials in SSA have shown OE is effective for palliation of KS symptoms, in patients without access to ART. OE PK exhibit high intersubject variability, and the dose-limiting side effect of OE, neutropenia, is related to etoposide exposure. The clinical risk of neutropenia is significantly higher in resource-limited settings, where access to supportive care may be limited. To date, the PK of OE in SSA populations has not been described; therefore the safety of OE is poorly defined. This trial describes first-dose PK of OE in Kenyan patients with HIV-related KS on ART. Methods: This clinical trial was IRB approved. HIV-positive adults (≥18 yo) with biopsy-proven KS, without prior KS therapy, but on ART were eligible for participation. Following informed consent, patients were given 50 mg oral etoposide (IV formulation), with collection of dried blood spots for subsequent PK analysis at pre-dose, 1, 2, 4, 6, and 8 hours post-dose. Following liquid extraction of the dried blood spots, etoposide concentrations were measured using a validated HPLC-MS/MS assay with teniposide as internal standard. Non-compartmental PK analysis was performed using the linear trapezoidal rule in MS-Excel. Results: 29 subjects participated in this clinical trial. Day 1 PK parameters are presented. Patients were on several different ART regimens, although none were on ritonavir, a known strong inhibitor of etoposide metabolism. PK Parameters of OE PK Parameter Mean Coefficient of Variation (CV%) Range Cmax (ng/mL) 611.7 36 114-1001 Tmax (h) 1.2 35 874-14760 AUC (0-inf) (ng/L.h) 3998 70 874-14760 CL/F (L/h) 18.25 64 3.39-57.21 Vd (L) 107 69 50-420 Conclusions: This study is the first to describe the PK of OE in Kenyans with HIV-associated KS on ART. While OE has exhibited high intersubject variability in prior studies, this study showed higher variability than previously reported: CV% in prior reports has generally been less than 50%, and in the present study is 60-70%. Ongoing analysis is exploring the role, if any, of body composition, co-medications, and genetics on the described PK variability. This preliminary data demonstrates that OE in a SSA population exhibits high intersubject variability, and therefore to ensure safety in this population, further study is needed of etoposide kinetics. Disclosure: All authors have declared no conflicts of interest.