Adverse event management in patients with advanced cancer receiving oral everolimus: focus on breast cancer.

BACKGROUND: Everolimus, an orally administered rapamycin analogue, inhibits the mammalian target of rapamycin (mTOR), a highly conserved intracellular serine-threonine kinase that is a central node in a network of signaling pathways controlling cellular metabolism, growth, survival, proliferation, angiogenesis, and immune function. Everolimus has demonstrated substantial clinical benefit in randomized, controlled, phase III studies leading to approval for the treatment of advanced renal cell carcinoma, advanced neuroendocrine tumors of pancreatic origin, renal angiomyolipoma and subependymal giant-cell astrocytoma associated with tuberous sclerosis complex, as well as advanced hormone-receptor-positive (HR(+)) and human epidermal growth factor receptor-2-negative advanced breast cancer. MATERIALS AND METHODS: We discuss clinically relevant everolimus-related adverse events from the phase III studies, including stomatitis, noninfectious pneumonitis, rash, selected metabolic abnormalities, and infections, with focus on appropriate clinical management of these events and specific considerations in patients with breast cancer. RESULTS: The majority of adverse events experienced during everolimus therapy are of mild to moderate severity. The safety profile and protocols for toxicity management are well established. The class-effect adverse event profile observed with everolimus plus endocrine therapy in breast cancer is (as expected) distinct from that of endocrine therapy alone, but is similar to that observed with everolimus in other solid tumors. Information gained from the experience in other carcinomas on prompt diagnosis and treatments to optimize drug exposure, treatment outcomes, and patients' quality of life also applies to the patient population with advanced breast cancer. CONCLUSIONS: As with all orally administered agents, education of both physicians and patients in the management of adverse events for patients receiving everolimus is critical to achieving optimal exposure and clinical benefit. Active monitoring for early identification of everolimus-related adverse events combined with aggressive and appropriate intervention should lead to a reduction in the severity and duration of the event.

Development and validation of a prognostic model in patients with metastatic renal cell carcinoma treated with sunitinib: a European collaboration.

BACKGROUND: Accurate prediction of outcome for metastatic renal cell carcinoma (mRCC) patients receiving targeted therapy is essential. Most of the available models have been developed in patients treated with cytokines, while most of them are fairly complex, including at least five factors. We developed and externally validated a simple model for overall survival (OS) in mRCC. We also studied the recently validated International Database Consortium (IDC) model in our data sets. METHODS: The development cohort included 170 mRCC patients treated with sunitinib. The final prognostic model was selected by uni- and multivariate Cox regression analyses. Risk groups were defined by the number of risk factors and by the 25th and 75th percentiles of the model's prognostic index distribution. The model was validated using an independent data set of 266 mRCC patients (validation cohort) treated with the same agent. RESULTS: Eastern Co-operative Oncology Group (ECOG) performance status (PS), time from diagnosis of RCC and number of metastatic sites were included in the final model. Median OS of patients with 1, 2 and 3 risk factors were: 24.7, 12.8 and 5.9 months, respectively, whereas median OS was not reached for patients with 0 risk factors. Concordance (C) index for internal validation was 0.712, whereas C-index for external validation was 0.634, due to differences in survival especially in poor-risk populations between the two cohorts. Predictive performance of the model was improved after recalibration. Application of the mRCC International Database Consortium (IDC) model resulted in a C-index of 0.574 in the development and 0.576 in the validation cohorts (lower than those recently reported for this model). Predictive ability was also improved after recalibration in this analysis. Risk stratification according to IDC model showed more similar outcomes across the development and validation cohorts compared with our model. CONCLUSION: Our model provides a simple prognostic tool in mRCC patients treated with a targeted agent. It had similar performance with the IDC model, which, however, produced more consistent survival results across the development and validation cohorts. The predictive ability of both models was lower than that suggested by internal validation (our model) or recent published data (IDC model), due to differences between observed and predicted survival among intermediate and poor-risk patients. Our results highlight the importance of external validation and the need for further refinement of existing prognostic models.

Maximising the duration of disease control in metastatic renal cell carcinoma with targeted agents: an expert agreement.

With six targeted agents approved (sorafenib, sunitinib, temsirolimus, bevacizumab [+interferon], everolimus and pazopanib), many patients with metastatic renal cell carcinoma (mRCC) will receive multiple therapies. However, the optimum sequencing approach has not been defined. A group of European experts reviewed available data and shared their clinical experience to compile an expert agreement on the sequential use of targeted agents in mRCC. To date, there are few prospective studies of sequential therapy. The mammalian target of rapamycin (mTOR) inhibitor everolimus was approved for use in patients who failed treatment with inhibitors of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) based on the results from a Phase III placebo-controlled study; however, until then, the only licensed agents across the spectrum of mRCC were VEGF(R) inhibitors (sorafenib, sunitinib and bevacizumab + interferon), and as such, a large body of evidence has accumulated regarding their use in sequence. Data show that sequential use of VEGF(R) inhibitors may be an effective treatment strategy to achieve prolonged clinical benefit. The optimal place of each targeted agent in the treatment sequence is still unclear, and data from large prospective studies are needed. The Phase III AXIS study of second-line sorafenib vs. axitinib (including post-VEGF(R) inhibitors) has completed, but the data are not yet published; other ongoing studies include the Phase III SWITCH study of sorafenib-sunitinib vs. sunitinib-sorafenib (NCT00732914); the Phase III 404 study of temsirolimus vs. sorafenib post-sunitinib (NCT00474786) and the Phase II RECORD 3 study of sunitinib-everolimus vs. everolimus-sunitinib (NCT00903175). Until additional data are available, consideration of patient response and tolerability to treatment may facilitate current decision-making regarding when to switch and which treatment to switch to in real-life clinical practice.

Targeted treatment for metastatic renal cell carcinoma and immune regulation.

New targeted agents have become the mainstream of treatment in metastatic renal cell carcinoma (mRCC) and substituted the previous cytokine-based therapies. Vascular endothelial growth factor (VEGF) pathway is the principle target for drugs like sunitinib, sorafenib and bevacizumab. As VEGF is regulating dendritic cell (DC) function, inhibition of VEGF results in activation of DCs and a shift towards cellular (type 1) immunity, which is believed to favor cancer rejection. Recent studies have established the immune-stimulating effects of sunitinib that may as well be a marker for effectiveness. On the other hand, sorafenib not only inhibits VEGF receptor (VEGFR) but is also a B-Raf inhibitor (a component of the ras - MAPK pathway) and this leads to downregulation of immune responses. Sorafenib has not yet shown benefit in first-line treatment of mRCC when compared to interferon (IFN)-alpha and sorafenib-mediated immunosuppression may partially account for that. Mammalian target of rapamycin (mTOR), the target of temsirolimus, is an element of the DC activation pathway. There are no data for in vivo effects of temsirolimus in the immune system. The addition of IFN-alpha to temsirolimus resulted in inferior outcomes than temsirolimus alone. IFN-alpha has however still a place in mRCC treatment, as bevacizumab has been approved in combination with IFN-alpha. New clinical trials address the effects of the combination of cytokines with targeted agents. The immune-modulating effects of targeted treatments may be important in pharmacodynamic outcomes, effectiveness or the development of adverse events.

Immunisation with 'naïve' syngeneic dendritic cells protects mice from tumour challenge.

Dendritic cells (DCs) 'pulsed' with an appropriate antigen may elicit an antitumour immune response in mouse models. However, while attempting to develop a DC immunotherapy protocol for the treatment of breast cancer based on the tumour-associated MUC1 glycoforms, we found that unpulsed DCs can affect tumour growth. Protection from RMA-MUC1 tumour challenge was achieved in C57Bl/6 MUC1 transgenic mice by immunising with syngeneic DCs pulsed with a MUC1 peptide. However, unpulsed DCs gave a similar level of protection, making it impossible to evaluate the effect of immunisation of mice with DCs pulsed with the specific peptide. Balb/C mice could also be protected from tumour challenge by immunisation with unpulsed DCs prior to challenge with murine mammary tumour cells (410.4) or these cells transfected with MUC1 (E3). Protection was achieved with as few as three injections of 50,000 naïve DCs per mouse per week, was not dependent on injection route, and was not specific to cell lines expressing human MUC1. However, the use of Rag2-knockout mice demonstrated that the adaptive immune response was required for tumour rejection. Injection of unpulsed DCs into mice bearing the E3 tumour slowed tumour growth. In vitro, production of IFN-gamma and IL-4 was increased in splenic cells isolated from mice immunised with DCs. Depleting CD4 T cells in vitro partially decreased cytokine production by splenocytes, but CD8 depletion had no effect. This paper shows that naïve syngeneic DCs may induce an antitumour immune response and has implications for DC immunotherapy preclinical and clinical trials.

The unexpected formation of biologically active Cu(II) Schiff mono-base complexes with 2-thiophene-carboxaldehyde and dipropylenetriamine: crystal and molecular structure of CudptaSCl2.

Two novel mononuclear Cu(II) coordination compounds of the type [Cu(dptaS)Cl(2)] and [Cu(dptaS)Br(2)] (dptaS=1,3-propanediamine, N(1)-[3-aminopropyl]-N(3)-[2-thienylmethylidene] or Schiff mono-base of dipropylenetriamine with 2-thiophene-carboxaldehyde) were prepared by the hydrolysis of the di-bases, [Cu(dptaSS)Cl(2)] and [Cu(dptaSS)Br(2)] (dptaSS=1,3-propanediamine, N(1)-[2-thienylmethylidene]-N(3)-[[2-thienylmethylidene]aminopropyl] or Schiff di-base of dipropylenetriamine with 2-thiophenecarboxaldehyde) to mono-bases with the release of one aldehyde molecule and freeing of the -NH(2) group of the coordinated dpta ligand. The X-ray determined structure of the compound [Cu(dptaS)Cl(2)] was confirmed by spectroscopic methods, magnetic and molar conductivity measurements. The Cu(II) atom is a five-coordinated CuN(3)Cl(2) chromophore with three nitrogen atoms coming up from the (dptaS) ligand and two chlorine atoms completing the square pyramidal geometry. Antiproliferative activity of both novel compounds was examined against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29 and T47D) and showed that the Cu(II) Schiff mono-bases exhibit increased activity as compared to the starting materials. In vitro studies of plasmid DNA (pDNA) and double stranded DNA (dsDNA) interaction with the compounds under study support this difference. Some of the important factors contributing to the antiproliferative activity of the compounds under study, such as ionic character and dipole moment were also discussed in terms of the density functional theory calculated electronic structures of the ligands and their Cu(II) compounds.

Safety and efficacy of the combination of trastuzumab with docetaxel for HER2-positive women with advanced breast cancer. A review of the existing clinical trials and results of the expanded access programme in the UK.

Trastuzumab is a humanised monoclonal antibody against the extracellular domain of HER2 (human epidermal growth factor receptor-2) that is overexpressed in about 25% of human breast cancers. It has shown clinical benefit in HER2-positive breast cancer cases when used alone or in combination with chemotherapy. Trastuzumab increases the response rate to chemotherapy and prolongs survival when used in combination with taxanes. In this article, we review the clinical trials where trastuzumab has been administered together with docetaxel, and we present the results of the trastuzumab expanded access programme (EAP) in the UK. Combination of trastuzumab with docetaxel results in similar response rates and time-to-progression with the trastuzumab/paclitaxel combinations. The toxicity of the combination and the risk of heart failure are low. The clinical data for the docetaxel/trastuzumab combination indicate a favourable profile from both the efficacy and the safety point of view and confirm the feasibility and safety of trastuzumab administration both as monotherapy and in combination with docetaxel.

Copper(II) Schiff base coordination compounds of dien with heterocyclic aldehydes and 2-amino-5-methyl-thiazole: synthesis, characterization, antiproliferative and antibacterial studies. Crystal structure of CudienOOCl2.

A new series of coordination compounds of the starting materials [Cu(dienX(2)Y(2))] and their adducts [Cu(dienXXY(2))(2a-5mt)] (where dien=diethylenetriamine, dienXX=Schiff bases of diethylenetriamine with 2-furaldehyde or 2-thiophene-carboxaldehyde, X=O, S, Y=Cl, Br, NO(3) and 2a-5mt=2-amino-5-methylthiazole) were synthesized by stepwise reactions and their structures were established by C, H, N, Cu analysis, spectroscopic, magnetic and molar conductivity measurements. The isolated compounds are monomers, paramagnetic and electrolytic compounds of the type 1:1. In all cases, the pentadentate Schiff base (dienXX) is bonded in a tridentate fashion through the 3 N atoms. In the CudienXXY(2) compounds the coordination sphere is completed by two Cl or Br or NO(3) groups in a square pyramidal arrangement. The proposed structure for this type of compound was further supported by X-ray diffraction analysis of the compound [Cu(dienOO)Cl(2)]. Its basal plane consists of three Cu-N contacts [2.017(2), 2.025(2) and 2.012(2) A] from dienOO, and the Cl(1) atom, while the Cl(2) atom possesses the apical position, the relevant distances being 2.2732(7) A for Cu-Cl(1) and 2.6051(7) A Cu-Cl(2). In the CudienX(2)Y(2).2a-5mt adducts the coordination sphere of copper is further completed by the nitrogen ring atom of the 2a-5mt, forming an octahedral configuration. The study of the biological activity of the compounds synthesized against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29, OAW42, T47D) and bacteria (E. coli, B. cereus, B. subtilis) showed that the adducts of the type [Cu(dienXXY(2))(2a-5mt)] exhibit increased activity both in cancer cells and in bacteria, compared to the starting material of type [Cu(dienXXY(2))].

Exercise-induced changes in c-Fos protein levels in skeletal muscle of trained and untrained rats.

We investigated the effect of an acute bout of endurance exercise on c-Fos protein levels in the extensor digitorum longus muscle of trained and untrained rats. Fifty rats were equally divided into a trained and an untrained group. Rats of the trained group ran on a treadmill 45 min/day for 5 days. On the sixth day, 5 rats were killed without exercise, while the remaining 20 ran as above and were killed 0, 3, 6, and 12 h post-exercise (5 rats at each time point). In the untrained group, 5 rats were killed without exercise, while the remaining 20 ran as above only once and were killed at the same time points as the trained group. Western blotting demonstrated no significant changes in c-Fos protein levels in the untrained group. On the contrary, in the trained group, there was a significant increase at 6 and 12 h compared to 3 h post-exercise. The levels of the protein in the trained rats were above the corresponding levels in the untrained ones at all time points, although these differences reached statistical significance only immediately, 6 h and 12 h post-exercise. These results show that trained skeletal muscle exhibits increased levels of c-Fos, probably as a cumulative result of changes occurring during recovery from each exercise bout, and greater c-Fos response after acute endurance exercise compared to untrained skeletal muscle.

Antiproliferative activity of mixed-ligand dien-Cu(II) complexes with thiazole, thiazoline and imidazole derivatives.

The reaction of [Cu(dien)NO(3)]NO(3) with 2-amino-5-methylthiazole (2A5MT), 2-amino-2-thiazoline (2A-2Tzn), imidazole (im), N,N'-thiocarbonyldiimidazole (Tcdim), 2-aminothiazole (2AT) and 2-ethylimidazole (2Etim), gave a new series of mixed-ligand compounds of the general formula [Cu(dien)(B)NO(3))]NO(3); (dien, diethylenetriamine; B, 2A5MT, 2A-2Tzn, im, Tcdim, 2AT and 2Etim). The complexes have been characterised by elemental analysis, molar conductivity and magnetic measurements, as well as by electronic and IR spectral studies. According to the above measurements the possible structure of the compounds is the square pyramidal in the solid state and the square planar in aqueous solution. We tested all complexes for antiproliferative (cytostatic and cytotoxic) activity against a panel of cell lines (HeLa, L929, HT-29 and T47D). All [(dien)Cu(B)NO(3))](NO(3)) complexes had an activity against colon cancer cells (HT-29), inducing G2/M cell cycle arrest, an effect that for most of the complexes could be attributed to p34cdc2 inhibition by tyrosine-phosphorylation and/or to induction of (cyclin-dependent kinase inhibitor) p21(WAF1). Other cell lines were resistant to the majority of the complexes, except [Cu(dien)(2A5MT)NO(3))](NO(3)), that had showed the highest anti-proliferative activity against HT-29 cells also. The predilection for colon cancer cells and the relatively low toxicity against normal (L929) cells justify further investigation of this group of compounds.

Antitumor activity of L-asparaginase from Thermus thermophilus.

L-asparaginase (EC 3.5.1.1) was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase was found to be 33 kDa by SDS-PAGE, whereas by Sephacryl S-300 superfine column it was found to be 200 kDa, indicating that the enzyme in the native stage acts as hexamer. It is a thermostable enzyme and keeps all of its activity at 80 degrees C for 10 min. The antiproliferative activity of the purified L-asparaginase from T. thermiphilus was tested against the following human cell lines: K-562 (chronic myelogenous leukemia), Raji (Burkitt's lymphoma), SK-N-MC (primitive neuroectodermal tumor), HeLa (cervical cancer), BT20 and MCF7 (breast cancers), HT-29 (human colon cancer), and OAW-42 (ovarian cancer). The antiproliferative activity of T. thermophilus enzyme was compared with Erwinase, the commercially available L-asparaginase from Erwinia corotovora. The potency difference between the two L-asparaginases was greater in HeLa and SK-N-MC than in other cell lines. The fact that L-asparaginase from T. thermophilus does not hydrolyse L-glutamine makes it advantageous for future clinical trials.

Protein tyrosine kinase inhibitor, genistein, enhances apoptosis and cell cycle arrest in K562 cells treated with gamma-irradiation.

Genistein, is a natural isoflavone compound with a potent activity against protein tyrosine kinases. The leukemic cell line, K562, is a bcr/abl (Philadelphia chromosome) positive cell line that is resistant to DNA-damaging agents, including gamma-irradiation. Treatment with genistein increased apoptosis and promoted G2-phase arrest in the non-apoptotic population of the gamma-irradiated K562 cells. Irradiated cells that survived 72 h after the irradiation had a normal distribution in cell cycle, whilst genistein treatment kept cells arrested in the G2-phase, decreased the S-phase fraction and suppressed DNA-synthesis. Taken together, our results show that genistein augments apoptotic cell death after gamma-irradiation in K562 cells and this result cannot be attributed to abrogation of the G2/M checkpoint.

CDK-inhibitor olomoucine inhibits cell death after exposure of cell lines to cytosine-arabinoside.

Signal transduction for apoptosis or programmed cell death, after DNA damage in mammalian cells, is believed to involve activation of cyclin-dependent kinases (CDKs), especially CDK-1 (cdc2) and CDK-2. We used CDK-inhibitor olomoucine, a purine analogue to evaluate the role CDK inhibition on cytosine-arabinoside (Ara-C)-induced cell death. The two drugs showed an antagonistic effect, suggesting that apoptosis after exposure to Ara-C is inhibited by olomoucine. DNA-electrophoresis showed a clear inhibition of the apoptotic pattern when olomoucine was added to Ara-C. We conclude that CDK-inhibitor olomoucine inhibits cell death induced by Ara-C.

Cytotoxicity of three resin-based root canal sealers: an in vitro evaluation.

The cytotoxicity of three resin-based root canal sealers (AH26, AH-Plus, Topseal) was evaluated in vitro. The experiments included two cell lines, L929 mouse skin fibroblasts and RPC-C2A rat pulp cells. The cytotoxicity was assessed by sulforodamine B (SRB) colorimetric assay and hemocytometer viable cell counting after 24- and 48-h exposure. AH26 had a severe cytotoxic effect whilst Topseal and AH-Plus showed a markedly lower toxic influence on the cells during the experimental period.

Antiproliferative activity of synthetic tetrapeptides, analogs of AS-I phytotoxin, towards cancer cell lines.

The in vitro chemosensitivity of three cancer cell lines [HT29 (colon), HeLa (cervical) and T47D (breast)] to eight synthetic tetrapeptides, analogs of AS-I toxin, with phytotoxic effect on a series of plants was studied. Mouse fibroblast L929 cell line was also tested for chemosensitivity to these peptides. All cell lines were especially sensitive to Cys-Val-Gly-Glu tetrapeptide with IC50 values of 0.18, 0.3 and 0.63 mM for HT29, HeLa and T47D cells, respectively, whereas the IC50 value for the L929 cells was higher than 1 mM. Antiproliferative activity was also observed with peptides Tyr-Val-Gly-Glu and His-Val-Gly-Glu with IC50 values higher than those obtained for Cys-Val-Gly-Glu. For the rest of the peptides tested the IC50 values were found close to or higher than 3 mM.

Effects of melatonin on proliferation of cancer cell lines.

The pineal hormone melatonin has been reported to have in vitro antiproliferative effects on estrogen receptor-positive human breast cancer cell lines at concentrations near to plasma physiological concentrations (1 x 10(-11) to 1 x 10(-9) M). Its growth inhibitory actions have been thought to be linked to the estrogen-receptor system. We tested the cytotoxic effects of melatonin on MCF-7 and T47D human breast cancer cell lines by using the SRB (sulforhodamine-B), XTT-tetrazolium, and bromodeoxyuridine (BrdU) assays in 96-well microtiter plates. After a 3 or 4 day exposure, melatonin did not have any significant effect on breast cancer cell proliferation and survival in doses up to 1 x 10(-4) M. Doses higher than 1 mM exhibited a potent cytotoxic effect, which was not mediated by the estrogen-receptor or by protein tyrosine kinases and was not specific for breast cancer cell lines. Intracellular glutathione levels did not seem to play any role in the sensitivity of breast cancer cells to melatonin, since the addition of L-buthionine-[S,R]-sulfoximine, ethacrynic acid, or exogenous glutathione did not modify our results. We conclude that under our experimental conditions melatonin has no inhibitory effects on human breast cancer cells at low (physiological or supraphysiological) concentrations. The different experimental procedures that were utilized in the present study can partially explain the divergence between our results and the literature.

Optimization of the sulforhodamine B colorimetric assay.

Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.