Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial.
BACKGROUND: In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. METHODS: In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. FINDINGS: 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17.9 months [IQR 10.3-29.2] in the control group vs 17.0 months [9.4-30.1] in the cetuximab group; HR 1.04, 95% CI 0.87-1.23, p=0.67). Similarly, there was no effect on progression-free survival (8.6 months [IQR 5.0-12.5] in the control group vs 8.6 months [5.1-13.8] in the cetuximab group; HR 0.96, 0.82-1.12, p=0.60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0.049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8.8 months (IQR 4.5-27.4); KRAS mutant, 14.4 months (8.5-24.0); all wild-type, 20.1 months (11.5-31.7). INTERPRETATION: This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended. FUNDING: Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA.
Intermittent versus continuous oxaliplatin and fluoropyrimidine combination chemotherapy for first-line treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial.
BACKGROUND: When cure is impossible, cancer treatment should focus on both length and quality of life. Maximisation of time without toxic effects could be one effective strategy to achieve both of these goals. The COIN trial assessed preplanned treatment holidays in advanced colorectal cancer to achieve this aim. METHODS: COIN was a randomised controlled trial in patients with previously untreated advanced colorectal cancer. Patients received either continuous oxaliplatin and fluoropyrimidine combination (arm A), continuous chemotherapy plus cetuximab (arm B), or intermittent (arm C) chemotherapy. In arms A and B, treatment continued until development of progressive disease, cumulative toxic effects, or the patient chose to stop. In arm C, patients who had not progressed at their 12-week scan started a chemotherapy-free interval until evidence of disease progression, when the same treatment was restarted. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and B is described in a companion paper. Here, we compare arms A and C, with the primary objective of establishing whether overall survival on intermittent therapy was non-inferior to that on continuous therapy, with a predefined non-inferiority boundary of 1.162. Intention-to-treat (ITT) and per-protocol analyses were done. This trial is registered, ISRCTN27286448. FINDINGS: 1630 patients were randomly assigned to treatment groups (815 to continuous and 815 to intermittent therapy). Median survival in the ITT population (n=815 in both groups) was 15.8 months (IQR 9.4-26.1) in arm A and 14.4 months (8.0-24.7) in arm C (hazard ratio [HR] 1.084, 80% CI 1.008-1.165). In the per-protocol population (arm A, n=467; arm C, n=511), median survival was 19.6 months (13.0-28.1) in arm A and 18.0 months (12.1-29.3) in arm C (HR 1.087, 0.986-1.198). The upper limits of CIs for HRs in both analyses were greater than the predefined non-inferiority boundary. Preplanned subgroup analyses in the per-protocol population showed that a raised baseline platelet count, defined as 400 000 per muL or higher (271 [28%] of 978 patients), was associated with poor survival with intermittent chemotherapy: the HR for comparison of arm C and arm A in patients with a normal platelet count was 0.96 (95% CI 0.80-1.15, p=0.66), versus 1.54 (1.17-2.03, p=0.0018) in patients with a raised platelet count (p=0.0027 for interaction). In the per-protocol population, more patients on continuous than on intermittent treatment had grade 3 or worse haematological toxic effects (72 [15%] vs 60 [12%]), whereas nausea and vomiting were more common on intermittent treatment (11 [2%] vs 43 [8%]). Grade 3 or worse peripheral neuropathy (126 [27%] vs 25 [5%]) and hand-foot syndrome (21 [4%] vs 15 [3%]) were more frequent on continuous than on intermittent treatment. INTERPRETATION: Although this trial did not show non-inferiority of intermittent compared with continuous chemotherapy for advanced colorectal cancer in terms of overall survival, chemotherapy-free intervals remain a treatment option for some patients with advanced colorectal cancer, offering reduced time on chemotherapy, reduced cumulative toxic effects, and improved quality of life. Subgroup analyses suggest that patients with normal baseline platelet counts could gain the benefits of intermittent chemotherapy without detriment in survival, whereas those with raised baseline platelet counts have impaired survival and quality of life with intermittent chemotherapy and should not receive a treatment break. FUNDING: Cancer Research UK.
ErbB-2 blockade and prenyltransferase inhibition alter epidermal growth factor and epidermal growth factor receptor trafficking and enhance (111)In-DTPA-hEGF Auger electron radiation therapy.
The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF. METHODS: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 x 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 x 10(6) EGFR per cell; 0.4 x 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 mug/mL) or L-778,123 (0-22.5 muM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123. RESULTS: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001). CONCLUSION: The therapeutic efficacy of (111)In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.
Chemotherapy options in elderly and frail patients with metastatic colorectal cancer (MRC FOCUS2): an open-label, randomised factorial trial.
BACKGROUND: Elderly and frail patients with cancer, although often treated with chemotherapy, are under-represented in clinical trials. We designed FOCUS2 to investigate reduced-dose chemotherapy options and to seek objective predictors of outcome in frail patients with advanced colorectal cancer. METHODS: We undertook an open, 2 x 2 factorial trial in 61 UK centres for patients with previously untreated advanced colorectal cancer who were considered unfit for full-dose chemotherapy. After comprehensive health assessment (CHA), patients were randomly assigned by minimisation to: 48-h intravenous fluorouracil with levofolinate (group A); oxaliplatin and fluorouracil (group B); capecitabine (group C); or oxaliplatin and capecitabine (group D). Treatment allocation was not masked. Starting doses were 80% of standard doses, with discretionary escalation to full dose after 6 weeks. The two primary outcome measures were: addition of oxaliplatin ([A vs B] + [C vs D]), assessed with progression-free survival (PFS); and substitution of fluorouracil with capecitabine ([A vs C] + [B vs D]), assessed by change from baseline to 12 weeks in global quality of life (QoL). Analysis was by intention to treat. Baseline clinical and CHA data were modelled against outcomes with a novel composite measure, overall treatment utility (OTU). This study is registered, number ISRCTN21221452. FINDINGS: 459 patients were randomly assigned (115 to each of groups A-C, 114 to group D). Factorial comparison of addition of oxaliplatin versus no addition suggested some improvement in PFS, but the finding was not significant (median 5.8 months [IQR 3.3-7.5] vs 4.5 months [2.8-6.4]; hazard ratio 0.84, 95% CI 0.69-1.01, p=0.07). Replacement of fluorouracil with capecitabine did not improve global QoL: 69 of 124 (56%) patients receiving fluorouracil reported improvement in global QoL compared with 69 of 123 (56%) receiving capecitabine. The risk of having any grade 3 or worse toxic effect was not significantly increased with oxaliplatin (83/219 [38%] vs 70/221 [32%]; p=0.17), but was higher with capecitabine than with fluorouracil (88/222 [40%] vs 65/218 [30%]; p=0.03). In multivariable analysis, fewer baseline symptoms (odds ratio 1.32, 95% CI 1.14-1.52), less widespread disease (1.51, 1.05-2.19), and use of oxaliplatin (0.57, 0.39-0.82) were predictive of better OTU. INTERPRETATION: FOCUS2 shows that with an appropriate design, including reduced starting doses of chemotherapy, frail and elderly patients can participate in a randomised controlled trial. On balance, a combination including oxaliplatin was preferable to single-agent fluoropyrimidines, although the primary endpoint of PFS was not met. Capecitabine did not improve QoL compared with fluorouracil. Comprehensive baseline assessment holds promise as an objective predictor of treatment benefit. FUNDING: Cancer Research UK and the Medical Research Council.
USP7 is involved in the cellular stress response by regulating Mdm2 and p53 protein levels following severe DNA damage. In addition to this, USP7 may also play a role in chromatin remodelling by direct deubiquitylation of histones, as well as indirectly by regulating the cellular levels of E3 ubiquitin ligases involved in histone ubiquitylation. Here, we provide new evidence that USP7 modulated chromatin remodelling is important for base excision repair of oxidative lesions. We show that transient USP7 siRNA knockdown did not change the levels or activity of base excision repair enzymes, but significantly reduced chromatin DNA accessibility and consequently the rate of repair of oxidative lesions. Scientific importance: The deubiquitylation enzyme USP7 was shown to stimulate the repair of oxidative DNA lesions by controlling histone ubiquitylation and therefore regulating accessibility of DNA repair enzymes to DNA damage.
The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.
USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase beta.
DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol beta) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol beta that is used as a source for nuclear Pol beta involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol beta, decreased levels of Pol beta, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity. Scientific importance:The deubiquitylation enzyme USP47 was identified as a regulator of the cellular levels of DNA polymerase beta in response to DNA damage.
Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells.
Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunitscontributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.
ZD6126 is a vascular-disrupting agent that affects the endothelial tubulin cytoskeleton causing selective occlusion of tumor vasculature and extensive tumor cell necrosis. The present study evaluated the antitumor and antivascular activities of ZD6126 in the clinically relevant murine renal cell carcinoma (RENCA) model and also evaluated biological response to therapy using color Doppler imaging as biomarker. Mice were implanted with RENCA tumor cells (day 0)and established tumors were treated with ZD6126 (100 mg/kg i.p.) or vehicle withrepeated intermittent doses on day 10, 14 and 18. ZD6126 treatment led to a significant reduction in tumor size and was associated with extensive tumor necrosis and a reduction in tumor blood flow versus controls. MVD increased withintermittent treatment (day 10, 14 and 18). In an additional study, animals weretreated at day 19 and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Corrosion casting showed that tumor vessel architecture is affected by treatment, whereas pre-existing vessels in control tissues are practically not affected. Inter-vessel and inter-branch distances as well as vessel diameters are influenced by treatment. In conclusion, ZD6126 showed potent antitumor efficacy in the RENCA model and our data suggest that decrease in tumor blood flow may bea useful surrogate marker of treatment effect.
Investigating temporal fluctuations in tumor vasculature with combined carbogen and ultrasmall superparamagnetic iron oxide particle (CUSPIO) imaging.
A combined carbogen ultrasmall superparamagnetic iron oxide (USPIO) imaging protocol was developed and applied in vivo in two murine colorectal tumor xenograft models, HCT116 and SW1222, with established disparate vascular morphology, to investigate whether additional information could be extracted from the combination of two susceptibility MRI biomarkers. Tumors were imaged before and during carbogen breathing and subsequently following intravenous administration of USPIO particles. A novel segmentation method was applied to the image data, from which six categories of R(2) * response were identified, and compared with histological analysis of the vasculature. In particular, a strong association between a negative DeltaR(2) *(carbogen) followed by positive DeltaR(2) *(USPIO) with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant DeltaR(2) *(carbogen) but no significant DeltaR(2) *(USPIO) were also identified, suggesting these regions became temporally isolated from the vascular supply during the experimental timecourse. These areas correlated with regions of tumortissue where there was CD31 staining but no Hoechst 33342 uptake. Significantly,different combined carbogen USPIO responses were determined between the two tumor models. Combining DeltaR(2) *(carbogen) and DeltaR(2) *(USPIO) with a novel segmentation scheme can facilitate the interpretation of susceptibility contrastMRI data and enable a deeper interrogation of tumor vascular function and architecture. Magn Reson Med, 2011. (c) 2011 Wiley-Liss, Inc.
Intercellular signalling plays an important role in the progression of a transformed cell to a tumour. In order to characterise the underlying mechanisms, a well-defined model cell system of intercellular induction of apoptosis was used where neighbouring normal cells can selectively eliminate transformed cells. In the absence of non-transformed cells, the induction of apoptosis in transformed 208Fsrc3 cells occurs via autocrine destruction and is dominated by peroxidase (PO), which initiates the PO/hypochlorous acid signalling pathway at high local cell densities. However, when the transformed cells are co-cultured with the non-transformed 208F cells, apoptosis in transformed cells additionally occurs as a result of intercellular signalling with the non-transformed cells and is predominantly due to the production of nitric oxide (NO(*)), which initiates the NO(*)/peroxynitrite pathway.
Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells.
Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that preventsactivating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find thatDRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (gammaH2AX). We accounted that about 72%of RAD51 foci co-localized with the observed gammaH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated withthe formation of a lesion that triggers RAD51-dependent homologous recombinationrepair, required for survival under transcriptional stress.
Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage.
The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster celllines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical gammaH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehydewas comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, gammaH2AX foci in FANCD1mt cells persisted for longertimes than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.
Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels andinduces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitorimpacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repairand may impact breast cancer therapy.
The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells.
Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptorknockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.
Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised (13)C MRS
Inflammation is a key component of many neurological diseases, yet our understanding of the contribution of these processes to tissue damage remains poor. For many such diseases, magnetic resonance imaging (MRI) has become the method of choice for clinical diagnosis. However, many of the MRI parameters that enable disease detection, such as passive contrast enhancement across a compromised blood-brain barrier, are weighted towards late-stage disease. Moreover, whilst these methods may report on disease severity, they are not able to provide information on either disease activity or the underlying molecular processes. There is a need, therefore, to develop methods that enable earlier disease detection, potentially long before clinical symptoms become apparent, together with identification of specific molecular processes that may guide specific therapy. This chapter describes the methodology for the synthesis and validation of two novel, functional MRI-detectable probes, based on microparticles of iron oxide (MPIO), which target endothelial adhesion molecules. These contrast agents enable the detection of acute brain inflammation in vivo, at a time when pathology is undetectable by conventional MRI. Such molecular MRI methods are opening new vistas for the acute diagnosis of CNS disease, together with the possibility for individually tailored therapy and earlier, more sensitive assessment of the efficacy of novel therapies.
The acute inflammatory response to intranigral alpha-synuclein differs significantly from intranigral lipopolysaccharide and is exacerbated by peripheral inflammation.
BACKGROUND: Activated microglia are a feature of the host response to neurodegeneration in Parkinson's disease (PD) and are thought to contribute to disease progression. Recent evidence suggests that extracellular alpha-synuclein (eSNCA) may play an important role in the pathogenesis of PD and that this may be mediated by a microglial response. METHODS: We wished to discover whether the host response to eSNCA would be sufficient to induce significant cytokine production. In vitro cultured BV-2 microglia were used to determine the basic inflammatory response to eSNCA. In vivo, 8-week old Biozzi mice were subjected to a single intranigral injection of either 3 mug SNCA, lipopolysaccharide (LPS) or serum protein (BSA) and allowed to recover for 24 hours. A second cohort of animals were peripherally challenged with LPS (0.5 mg/kg) 6 hours prior to tissue collection. Inflammation was studied by quantitative real-time PCR for a number of pro-inflammatory genes and immunohistochemistry for microglial activation, endothelial activation and cell death. RESULTS: In vitro data showed a robust microglial response to SNCA, including a positive NFkB response and the production of pro-inflammatory cytokines. Direct injection of SNCA into the substantia nigra resulted in the upregulation of mRNA expression of proinflammatory cytokines, the expression of endothelial markers of inflammation and microglial activation. However, these results were significantly different to those obtained after direct injection of LPS. By contrast, when the animals were injected intracerebrally with SNCA and subsequently challenged with systemic LPS, the level of production of IL-1beta in the substantia nigra became comparable to that induced by the direct injection of LPS into the brain. The injection of albumin into the nigra with a peripheral LPS challenge did not provoke the production of a significant inflammatory response. Direct injection of LPS into the substantia nigra also induces cell death in a more robust manner than direct injection of either SNCA or BSA. CONCLUSION: These results suggest that the presence of eSNCA protein 'primes' microglia, making them susceptible to environmental proinflammatory challenge. For this reason, we hypothesise that where 'inflammation' contributes to the disease progression in PD, it does so in a punctuate manner (on-off) as a result of systemic events.
Phase II study of conformal hypofractionated radiotherapy with concurrent gemcitabine in muscle-invasive bladder cancer.
PURPOSE The aim of this prospective, phase II trial was to determine the response of muscle-invasive bladder cancer (MIBC) to concurrent chemoradiotherapy of weekly gemcitabine with 4 weeks of radiotherapy (RT; GemX). PATIENTS AND METHODS. Fifty patients with transitional cell carcinoma, stage T2-3, N0, M0 after transurethral resection and magnetic resonance imaging, were recruited. Gemcitabine was given intravenously at 100 mg/m(2) on days 1, 8, 15, and 22 of a28-day RT schedule that delivered 52.5 Gy in 20 fractions. Chemotherapy was stopped for Radiation Therapy Oncology Group (RTOG) grade 3 bladder or bowel toxicity. The primary end points were tumor response, toxicity, and survival. Results All patients completed RT; 46 tolerated all four cycles of gemcitabine. Two patients stopped after two cycles, and two stopped after three cycles, because of bowel toxicity. Forty-seven patients had a post-treatment cystoscopy;44 (88%) achieved a complete endoscopic response. At a median follow-up of 36 months (range, 15 to 62 months), 36 patients were alive, and 32 of these had a functional and intact bladder. Fourteen patients died; seven died as a result ofmetastatic MIBC, five died as a result of intercurrent disease, and two died as a result of treatment-associated deaths. Four patients underwent cystectomy; threebecause of recurrent disease and one because of toxicity. One patient required abowel resection for late toxicity. By using Kaplan-Meier analyses, 3-year cancer-specific survival was 82%, and overall survival was 75%. CONCLUSION Concurrent gemcitabine-based chemoradiotherapy (ie, GemX) produces a high response rate in MIBC and has durable local control and acceptable toxicity, which allows patients to preserve their own bladder. This treatment modality warrants additional investigation in a phase III setting.
Human AlkB homologue 5 is a nuclear 2-oxoglutarate dependent oxygenase and a direct target of hypoxia-inducible factor 1alpha (HIF-1alpha).
Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.